Closed-loop biochemical analyzers

ABSTRACT

Integrated systems, apparatus, software, and methods for performing biochemical analysis, including DNA sequencing, genomic screening, purification of nucleic acids and other biological components and drug screening are provided. Microfluidic devices, systems and methods for using these devices and systems for performing a wide variety of fluid operations are provided. The devices and systems of are used in performing fluid operations which require a large number of iterative, successive or parallel fluid manipulations, in a microscale, or sealed and readily automated format.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of provisional patentapplication U.S. Ser. No. 60/068,311, entitled “Closed Loop BiochemicalAnalyzer” by Knapp, filed Dec. 19, 1997. The subject application is alsoa continuation-in-part of 08/835,101 by Knapp et al. filed Apr. 4, 1997,abandoned, converted to a provisional application 60/086,240 filed Apr.4,1997 by filing a petition under 37 C.F.R. §§1.53(C) and 1.17(a) onJan. 20, 1998), entitled “Microfluidic Devices and Systems forPerforming Integrated Fluid Operations.” Both of these applications areincorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This application relates to apparatus, methods and integrated systemsfor detecting molecular interactions. The apparatus comprise microscaledevices for moving and mixing small fluid volumes. The systems arecapable of performing integrated manipulation and analysis in a varietyof biological, biochemical and chemical experiments, including, e.g.,DNA sequencing.

BACKGROUND OF THE INVENTION

Manipulating fluidic reagents and assessing the results of reagentinteractions are central to chemical and biological science.Manipulations include mixing fluidic reagents, assaying productsresulting from such mixtures, and separation or purification of productsor reagents and the like. Assessing the results of reagent interactionscan include autoradiography, spectroscopy, microscopy, photography, massspectrometry, nuclear magnetic resonance and many other techniques forobserving and recording the results of mixing reagents. A singleexperiment can involve literally hundreds of fluidic manipulations,product separations, result recording processes and data compilation andintegration steps. Fluidic manipulations are performed using a widevariety of laboratory equipment, including various fluid heatingdevices, fluidic mixing devices, centrifugation equipment, moleculepurification apparatus, chromatographic machinery, gel electrophoreticequipment and the like. The effects of mixing fluidic reagents aretypically assessed by additional equipment relating to detection,visualization or recording of an event to be assayed, such asspectrophotometers, autoradiographic equipment, microscopes, gelscanners, computers and the like.

Because analysis of even simple chemical, biochemical, or biologicalphenomena requires many different types of laboratory equipment, themodern laboratory is complex, large and expensive. In addition, becauseso many different types of equipment are used in even conceptuallysimple experiments such as DNA sequencing, it has not generally beenpractical to integrate different types of equipment to improveautomation. The need for a laboratory worker to physically perform manyaspects of laboratory science imposes sharp limits on the number ofexperiments which a laboratory can perform, and increases theundesirable exposure of laboratory workers to toxic or radioactivereagents. In addition, results are often analyzed manually, with theselection of subsequent experiments related to initial experimentsrequiring consideration by a laboratory worker, severely limiting thethroughput of even repetitive experimentation.

In an attempt to increase laboratory throughput and to decrease exposureof laboratory workers to reagents, various strategies have beenperformed. For example, robotic introduction of fluids onto microtiterplates is commonly performed to speed mixing of reagents and to enhanceexperimental throughput. More recently, microscale devices for highthroughput mixing and assaying of small fluid volumes have beendeveloped. For example, U.S. Ser. No. 08/761,575 now U.S. Pat. No.6,046,056 entitled “High Throughput Screening Assay Systems inMicroscale Fluidic Devices” by Parce et al. provides pioneeringtechnology related to microscale fluidic devices, especially includingelectrokinetic devices. The devices are generally suitable for assaysrelating to the interaction of biological and chemical species,including enzymes and substrates, ligands and ligand binders, receptorsand ligands, antibodies and antibody ligands, as well as many otherassays. Because the devices provide the ability to mix fluidic reagentsand assay mixing results in a single continuous process, and becauseminute amounts of reagents can be assayed, these microscale devicesrepresent a fundamental advance for laboratory science.

In the electrokinetic microscale devices provided by Parce et al. above,an appropriate fluid is flowed into a microchannel etched in a substratehaving functional groups present at the surface. The groups ionize whenthe surface is contacted with an aqueous solution. For example, wherethe surface of the channel includes hydroxyl functional groups at thesurface, e.g., as in glass substrates, protons can leave the surface ofthe channel and enter the fluid. Under such conditions, the surfacepossesses a net negative charge, whereas the fluid will possess anexcess of protons, or positive charge, particularly localized near theinterface between the channel surface and the fluid. By applying anelectric field along the length of the channel, cations will flow towardthe negative electrode. Movement of the sheath of positively chargedspecies in the fluid pulls the solvent with them.

One time consuming process is titration of biological and biochemicalassay components into the dynamic range of an assay. For example,because enzyme activities vary from lot to lot, it is necessary toperform a titration of enzyme and substrate concentrations to determineoptimum reaction conditions. Similarly, diagnostic assays requiretitration of unknown concentrations of components so that the assay canbe performed using appropriate concentrations of components. Thus, evenbefore performing a typical diagnostic assay, several normalizationsteps need to be performed with assay components.

Another labor intensive laboratory process is the selection of leadcompounds in drug screening assays. Various approaches to screening forlead compounds are reviewed by Janda (1994) Proc. Natl. Acad. Sci. USA91(10779-10785); Blondelle (1995) Trends Anal. Chem 14:83-91; Chen etal. (1995) Angl. Chem. Int. Engl. 34:953-960; Ecker et al. (1995)Bio/Technology 13:351-360; Gordon et al. (1994) J. Med. Chem.37:1385-1401 and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.Improvements in screening have been developed by combining one or moresteps in the screening process, e.g., affinity capillaryelectrophoresis-mass spectrometry for combinatorial library screening(Chu et al. (1996) J. Am. Chem. Soc. 118:7827-7835). However, thesehigh-throughput screening methods do not provide an integrated way ofselecting a second assay or screen based upon the results of a firstassay or screen. Thus, results from one assay are not automatically usedto focus subsequent experimentation and experimental design stillrequires a large input of labor by the user.

Another particularly labor intensive biochemical series of laboratoryfluidic manipulations is nucleic acid sequencing. Efficient DNAsequencing technology is central to the development of the biotechnologyindustry and basic biological research. Improvements in the efficiencyand speed of DNA sequencing are needed to keep pace with the demands forDNA sequence information. The Human Genome Project, for example, has seta goal of dramatically increasing the efficiency, cost-effectiveness andthroughput of DNA sequencing techniques. See, e.g., Collins, and Galas(1993) Science 262:43-46.

Most DNA sequencing today is carried out by chain termination methods ofDNA sequencing. The most popular chain termination methods of DNAsequencing are variants of the dideoxynucleotide mediated chaintermination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad.Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing,see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (Supplement 37, current through 1997)(Ausubel), Chapter 7. Four color sequencing is described in U.S. Pat.No. 5,171,534. Thousands of laboratories employ dideoxynucleotide chaintermination techniques. Commercial kits containing the reagents mosttypically used for these methods of DNA sequencing are available andwidely used.

In addition to the Sanger methods of chain termination, new PCRexonuclease digestion methods have also been proposed for DNAsequencing. Direct sequencing of PCR generated amplicons by selectivelyincorporating boronated nuclease resistant nucleotides into theamplicons during PCR and digestion of the amplicons with a nuclease toproduce sized template fragments has been proposed (Porter et al. (1997)Nucleic Acids Research 25(8):1611-1617). In the methods, 4 PCR reactionson a template are performed, in each of which one of the nucleotidetriphosphates in the PCR reaction mixture is partially substituted witha 2′deoxynucleoside 5′-α[P-borano]-triphosphate. The boronatednucleotide is stocastically incorporated into PCR products at varyingpositions along the PCR amplicon in a nested set of PCR fragments of thetemplate. An exonuclease which is blocked by incorporated boronatednucleotides is used to cleave the PCR amplicons. The cleaved ampliconsare then separated by size using polyacrylamide gel electrophoresis,providing the sequence of the amplicon. An advantage of this method isthat it requires fewer biochemical manipulations than performingstandard Sanger-style sequencing of PCR amplicons.

Other sequencing methods which reduce the number of steps necessary fortemplate preparation and primer selection have been developed. Oneproposed variation on sequencing technology involves the use of modularprimers for use in PCR and DNA sequencing. For example, Ulanovsky andco-workers have described the mechanism of the modular primer effect(Beskin et al. (1995) Nucleic Acids Research 23(15):2881-2885) in whichshort primers of 5-6 nucleotides can specifically prime atemplate-dependent polymerase enzyme for template dependent nucleic acidsynthesis. A modified version of the use of the modular primer strategy,in which small nucleotide primers are specifically elongated for use inPCR to amplify and sequence template nucleic acids has also beendescribed. The procedure is referred to as DNA sequencing usingdifferential extension with nucleotide subsets (DENS). See, Raja et al.(1997) Nucleic Acids Research 25(4):800-805.

In addition to enzymatic and other chain termination sequencing methods,sequencing by hybridization to complementary oligonucleotides has beenproposed, e.g., in U.S. Pat. No. 5,202,231, to Drmanac et al. and, e.g.,in Drmanac et al. (1989) Genomics 4:114-128. Chemical degradationsequencing methods are also well known and still in use; see, Maxam andGilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York,Methods in Enzymology 65:499-560.

Improvements in methods for generating sequencing templates have alsobeen developed. DNA sequencing typically involves three steps: i) makingsuitable templates for the regions to be sequenced; ii) runningsequencing reactions for electrophoresis and iii) assessing the resultsof the reaction. The latter steps are sometimes automated by use oflarge and very expensive workstations and autosequencers. The first stepoften requires careful experimental design and laborious DNAmanipulation such as the construction of nested deletion mutants. See,Griffin, H. G. and Griffin, A. M. (1993) DNA sequencing protocols,Humana Press, New Jersey. Alternatively, random “shot-gun” sequencingmethods, are sometimes used to make templates, in which randomlyselected sub clones, which may or may not have overlapping sequenceinformation, are randomly sequenced. The sequences of the sub clones arecompiled to produce an ordered sequence. This procedures eliminatescomplicated DNA manipulations; however, the method is inherentlyinefficient because many recombinant clones must be sequenced due to therandom nature of the procedure. Because of the labor intensive nature ofsequencing, the repetitive sequencing of many individual clonesdramatically reduces the throughput of these sequencing systems.

Recently, Hagiwara and Curtis (1996) Nucleic Acids Research24(12):2460-2461 developed a “long distance sequencer” PCR protocol forgenerating; overlapping nucleic acids from very large clones tofacilitate sequencing, and methods of amplifying and tagging theoverlapping nucleic acids into suitable sequencing templates. Themethods can be used in conjunction with shotgun sequencing techniques toimprove the efficiency of shotgun methods.

Although improvements in robotic manipulation of fluidic reagents andminiaturization of laboratory equipment have been made, and althoughparticular biochemical processes such as DNA sequencing and drugscreening are very well developed, there still exists a need foradditional techniques and apparatus for mixing and assaying fluidicreagents, for integration of such systems and for reduction of thenumber of manipulations required to perform biochemical manipulationssuch as drug screening and DNA sequencing. Ideally, these new apparatuswould be useful with, and compatible to, established biochemicalprotocols. This invention provides these and many other features.

SUMMARY OF THE INVENTION

This invention provides apparatus, systems and methods for integratedmanipulation and analysis of fluidic reagents. The integrated featuresprovide very high throughput methods of assessing biochemical componentsand performing biochemical manipulations. A wide variety of reagents andproducts are suitably assessed, including libraries of chemical orbiological compounds or components, nucleic acid templates, PCR reactionproducts, and the like. In the integrated systems it is possible to usethe results of a first reaction or set of reactions to selectappropriate reagents, reactants, products, or the like, for additionalanalysis. For example, the results of a first sequencing reaction can beused to select primers, templates or the like for additional sequencing,or to select related families of compounds for screening inhigh-throughput assay methods. These primers or templates are thenaccessed by the system and the process continues.

In one aspect, the invention provides integrated methods of analyzingand manipulating sample materials for fluidic analysis. In the methods,an integrated microfluidic system including a microfluidic device isprovided. The device has at least a first reaction channel and at leasta first reagent introduction channel, typically etched, machined,printed, or otherwise manufactured in or on a substrate. Optionally, thedevice can have a second reaction channel and/or reagent introductionchannel, a third reaction channel and/or reagent introduction channel orthe like, up to and including hundreds or even thousands of reactionand/or reagent introduction channels. The reaction channel and reagentintroduction channels are in fluid communication, i.e., fluid can flowbetween the channels under selected conditions. The device has amaterial transport system for controllably transporting a materialthrough and among the reagent introduction channel and reaction channel.For example, the material transport system can include electrokinetic,electroosmotic, electrophoretic or other fluid manipulation aspects(micro-pumps and microvalves, fluid switches, fluid gates, etc.) whichpermit controlled movement and mixing of fluids. The device also has afluidic interface in fluid communication with the reagent introductionchannel. Such fluidic interfaces optionally include capillaries,channels, pins, pipettors, electropipettors, or the like, for movingfluids, and optionally further include microscopic, spectroscopic, fluidseparatory or other aspects. The fluidic interface samples a pluralityof reagents or mixtures of reagents from a plurality of sources ofreagents or mixtures of reagents and introduces the reagents or mixturesof reagents into the reagent introduction channel. Essentially anynumber of reagents or reagent mixtures can be introduced by the fluidicinterface, depending on the desired application. Because microfluidicmanipulations are performed in a partially or fully sealed environment,contamination and fluidic evaporation in the systems are minimized.

In the methods, a first reagent from the plurality of sources of reagentor mixtures of reagents is selected. A first sample material and thefirst reagent or mixture of reagents is introduced into the firstreaction channel, whereupon the first sample material and the firstreagent or mixture of reagents react. This reaction can take a varietyof different forms depending on the nature of the reagents. For example,where the reagents bind to one another, such as where the reagents arean antibody or cell receptor and a ligand, or an amino acid and abinding ligand, the reaction results in a bound component such as abound ligand. Where the reagents are sequencing reagents, a primerextension product results from the reaction. Where the reagents includeenzymes and enzyme substrates, a modified form of the substratetypically results. Where two reacting chemical reagents are mixed, athird product chemical typically results.

In the methods, a reaction product of the first sample material and thefirst reagent or mixture of reagents is analyzed. This analysis can takeany of a variety of forms, depending on the application. For example,where the product is a primer extension product, the analysis can takethe form of separating reactants by size, detecting the sized reactantsand translating the resulting information to give the sequence of atemplate nucleic acid. Similarly, because microscale fluidic devices ofthe invention are optionally suitable for heating and cooling areaction, a PCR reaction utilizing PCR reagents (thermostablepolymerase, nucleotides, templates, primers, buffers and the like) canbe performed and the PCR reagents detected. Where the reaction resultsin the formation of a new product, such as an enzyme-substrate product,a chemical species, or an immunological component such as a boundligand, the product is typically detected by any of a variety ofdetection techniques, including autoradiography, microscopy,spectroscopy, or the like.

Based upon the reaction product, a second reagent or mixture of reagentsis selected and a second sample material is assessed. For example, wherethe product is a DNA sequence, a sequencing primer and/or template forextension of available sequence information is selected. Where theproduct is a new product such as those above, an appropriate secondcomponent such as an enzyme, ligand, antibody, receptor molecule,chemical, or the like, is selected to further test the binding orreactive characteristics of an analyzed material. The second reagent ormixture of reagents is introduced into the first reaction channel, oroptionally into a second (or third or fourth . . . or nth) reactionchannel in the microfluidic device. The second sample material and thesecond reagent or mixture of reagents react, forming a new product,which is analyzed as above. The results of the analysis can serve as thebasis for the selection and analysis of additional reactants for similarsubsequent analysis. The second sample material, reagents, or mixturesof reagents can comprise the same or different materials. For example, asingle type of DNA template is optionally sequenced in several serialreactions. Alternatively, completing a first sequencing reaction, asoutlined above, serves as the basis for selecting additional templates(e.g., overlapping clones, PCR amplicons, or the like).

Accordingly, in a preferred aspect, the invention provides methods ofsequencing a nucleic acid. In the methods, the biochemical components ofa sequencing reaction (e.g., a target nucleic acid, a first andoptionally, second sequencing primer, a polymerase (optionally includingthermostable polymerases for use in PCR), dNTPs, and ddNTPs) are mixedin a microfluidic device under conditions permitting target dependentpolymerization of the dNTPs. Polymerization products are separated inthe microfluidic device to provide a sequence of the target nucleicacid. Typically, sequencing information acquired by this method is usedto select additional sequencing primers and/or templates, and theprocess is reiterated. Generally, a second sequencing primer is selectedbased upon the sequence of the target nucleic acid and the secondsequencing primer is mixed with the target nucleic acid in amicrofluidic device under conditions permitting target dependentelongation of the selected second sequencing primer, thereby providingpolymerization products which are separated by size in the microfluidicdevice to provide further sequence of the target nucleic acid. Asdiscussed above, the systems for mixing the biochemical sequencingcomponents, separating the reaction products, and assessing the resultsof the sequencing reaction are integrated into a single system.

In one integrated sequencing system, methods of sequencing a targetnucleic acid are provided in which an integrated microfluidic systemcomprising a microfluidic device is utilized in the sequencing method.The integrated microfluidic device has at least a first sequencingreaction channel and at least a first sequencing reagent introductionchannel, the sequencing reaction channel and sequencing reagentintroduction channel being in fluid communication. The integratedmicrofluidic system also has a material transport system forcontrollably transporting sequencing reagents through the sequencingreagent introduction channel and sequencing reaction channel and afluidic interface in fluid communication with the sequencing reagentintroduction channel for sampling a plurality of sequencing reagents, ormixtures of sequencing reagents, from a plurality of sources ofsequencing reagents or mixtures of sequencing reagents and introducingthe sequencing reagents or mixtures of sequencing reagents into thesequence reagent introduction channel. As discussed above, the interfaceoptionally includes capillaries, pins, pipettors and the like. In themethod, a first sequencing primer sequence complementary to a firstsubsequence of a first target nucleic acid sequence is introduced intothe sequence reagent introduction channel. The first primer ishybridized to the first subsequence and the first primer is extendedwith a polymerase enzyme along the length of the target nucleic acidsequence to form a first extension product that is complementary to thefirst subsequence and a second subsequence of the target nucleic acid.The sequence of the first extension product is determined and, basedupon the sequence of the first extension product, a second primersequence complementary to a second subsequence of the target nucleicacid sequence is selected, hybridized and extended as above.

In the sequence methods herein, it is sometimes advantageous to haveselect sequencing primers from a large set of sequencing primers, ratherthan synthesizing primers to match a particular target nucleic acid. Forexample, 5 or 6-mer primers can be made to hybridize specifically to atarget, e.g., where the primers are modular and hybridize to a singleregion of a nucleic acid. All possible 5 or 6 mers can be synthesizedfor selection in the methods herein, or any subset of 5 or 6 mers canalso be selected. In some embodiments, the primers are transferred tothe microfluidic apparatus, e.g., by a capillary, an electropipettor, orusing sipping technology, from a microtiter plate or from and array ofoligos. In other embodiments, the primers are located on a region of amicrofluidic device, chip or other substrate.

An advantage of these sequencing methods is that they dramaticallyincrease the speed with which sequencing reactions can be performed. Anentire sequencing reaction, separation of sequencing products andsequence generation can be performed in less than an hour, often lessthan 30 minutes, generally less than 15 minutes, sometimes less than 10minutes and occasionally less than 5 minutes.

The present invention provides integrated systems and apparatus forperforming the sequencing methods herein. In one embodiment, theinvention provides a sequencing apparatus. The apparatus has a topportion, a bottom portion and an interior portion. The interior portionhas at least two intersecting channels (and often tens, hundreds, orthousands of intersecting channels), wherein at least one of the twointersecting channels has at least one cross sectional dimension betweenabout 0.1 μm and 500 μm. A preferred embodiment of the inventionincludes an electrokinetic fluid direction system for moving asequencing reagent through at least one of the two intersectingchannels. The apparatus further includes a mixing zone fluidly connectedto the at least two intersecting channels for mixing the sequencingreagents, and a size separation zone fluidly connected to the mixingzone for separating sequencing products by size, thereby providing thesequence of a target nucleic acid. Optionally, the apparatus has asequence detector for reading the sequence of the target nucleic acid.In one preferred embodiment, the apparatus has a set of wells forreceiving reagents such as primer sets for use in the apparatus. In oneembodiment, the apparatus has at least 4,096 wells fluidly connected tothe at least two intersecting channels. Alternatively, the apparatus caninclude a substrate (matrix, or membrane) with primers located on thesubstrate. Often, the primers will be dried in spots on the substrate.In this embodiment, the apparatus will typically include anelectropipettor which has a tip designed to re-hydrate a selected spotcorresponding to a dried primer, and for electrophoretic transport ofthe rehydrated primer to an analysis region in the microfluidic device(i.e., a component of the microfluidic device which includes a reactionchannel). Thus, in a preferred embodiment, the device will include asubstrate such as a membrane having, e.g., 4,096 spots (i.e., allpossible 6-mer primers). Similarly, components in diagnostic or drugscreening assays can be stored in the well or membrane format forintroduction into the analysis region of the device. Arrays of nucleicacids, proteins and other compounds are also used in a similar manner.

In another embodiment, the invention provides systems for determining asequence of nucleotides in a target nucleic acid sequence. The systemincludes a microfluidic device having a body structure with at least afirst mixing or analysis channel, and at least a first probeintroduction channel disposed therein, the analysis channel intersectingand being in fluid communication with the probe introduction channel.The system includes a source of the target nucleic acid sequence influid communication with the analysis channel and a plurality ofseparate sources of oligonucleotide probes in fluid communication withthe probe introduction channel, each of the plurality of separatesources containing an oligonucleotide probe having a differentnucleotide sequence of length n. Typically, all or essentially all(i.e., most, i.e., at least about 70%, typically 90% or more) of thepossible oligonucleotides of a given length are included, although asubset of all possible oligonucleotides can also be used. The systemalso includes a sampling system for separately transporting a volume ofeach of the oligonucleotide probes from the sources of oligonucleotideprobes to the probe introduction channel and injecting each of theoligonucleotide probes into the analysis channel to contact the targetnucleic acid sequence and a detection system for identifying whethereach oligonucleotide probe hybridizes with the target nucleic acidsequence.

Methods of using the system for sequencing by hybridization to perfectlymatched probes are also provided. In these methods, a target nucleicacid is flowed into the analysis channel and a plurality of extensionprobes are separately injected into the analysis channel, whereupon theextension probes contact the target nucleic acid sequence. In themethod, a first subsequence of nucleotides in the target nucleic acid istypically known, and each of the plurality of extension probes has afirst sequence portion that is perfectly complementary to at least aportion of the first subsequence, and an extension portion thatcorresponds to a portion of the target nucleic acid sequence adjacent tothe target subsequence, the extension portion having a length n, andcomprising all possible nucleotide sequences of length n, wherein n isbetween 1 and 4 inclusive. A sequence of nucleotides is identifiedadjacent the target subsequence, based upon which of the plurality ofextension probes perfectly hybridizes with the target nucleic acidsequence.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts a graph of fluorescence signal of intercalating dye forlambda genomic DNA.

FIG. 2 depicts a thermocycler channel with varying widths forperforming, e.g., PCR.

FIG. 3 depicts a top view of a non-thermal amplification apparatus.

FIGS. 4A-4D depicts a top view of a reaction and separation apparatusand output data from the apparatus.

FIG. 5A depicts a top view of an apparatus for discriminating nucleicacids based on sequencing; FIG. 5B depicts an optional separationchannel.

FIGS. 6A-6B depict alternate technologies for flowing dried reagentsfrom a substrate into a microfluidic apparatus; 6A depicts anelectropipettor with a cup region; 6B depicts an electrokineticinterface which spans a membrane having the dried reagents.

FIGS. 7A-7D depict serial to parallel conversion strategies.

FIG. 8 depicts a top view of a serial to parallel converter.

FIG. 9 depicts a top view of a serial to parallel converter.

FIG. 10 depicts a top view of a serial to parallel converter.

FIG. 11 depicts a top view of a serial to parallel converter.

FIG. 12 depicts a block diagram of a control system as connected to amicrofluidic device.

FIG. 13 is a top view of an integrated microfluidic device having astorage substrate in the same plane as an analysis substrate.

FIG. 14 is a top view of an integrated microfluidic devices having astorage substrate in a plane different from an analysis substrate.

FIG. 15 is a top view of a microfluidic substrate having an integratedelectropipettor.

FIG. 16 is a top view of a microfluidic substrate having an integratedelectropipettor in the form of a capillary tube.

FIG. 17 is a top view of a microfluidic substrate having an integratedelectropipettor with serpentine channel geometry useful forelectrophoresis.

FIG. 18 is a top view of an integrated microfluidic device incorporatinga microtiter dish.

FIG. 19 is a flowchart outlining some of the software processing stepsperformed by a computer in an integrated system of the invention.

FIG. 20 is a schematic of an integrated system for sequencing nucleicacids.

FIG. 21 is a top view of a microchip of the invention.

FIG. 22 is an electropherogram for an assay.

FIG. 23 is an electropherogram for an assay in which white blood cellsare electrophoresed.

DEFINITIONS

An “integrated microfluidic system” is a microfluidic system in which aplurality of fluidic operations are performed. In one embodiment, theresults of a first reaction in the microfluidic system are used toselect reactants or other reagents for a second reaction or assay. Thesystem will typically include a microfluidic substrate, and a fluidicinterface for sampling reactants or other components. A detector and acomputer are often included for detecting reaction products and forrecording, selecting, facilitating and monitoring reactions in themicrofluidic substrate.

A “microfluidic device” is an apparatus or component of an apparatushaving microfluidic reaction channels and/or chambers. Typically, atleast one reaction channel or chamber will have at least onecross-sectional dimension between about 0.1 μm and about 500 μm.

A “reaction channel” is a channel (in any form, including a closedchannel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) in which two or more components are mixed. Thechannel will have at least one region with a cross sectional dimensionof between about 0.1 μm and about 500 μm.

A “reagent channel” is a channel (in any form, including a closedchannel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) through which components are transported(typically suspended or dissolved in a fluid). The channel will have atleast one region with a cross sectional dimension of between about 0.1μm and about 500 μm.

A “material transport system” is a system for moving components along orthrough microfluidic channels. Exemplar transport systems includeelectrokinetic, electroosmotic, and electrophoretic systems (e.g.,electrodes in fluidly connected wells having a coupled current and/orvoltage controller), as well as micro-pump and valve systems.

A “fluidic interface” in the context of a microfluidic substrate is acomponent for transporting materials into or out of the substrate. Theinterface can include, e.g., an electropipettor, capillaries, channels,pins, pipettors, sippers or the like for moving fluids into themicrofluidic substrate.

The overall function, i.e., intended goal, of the devices, systems andmethods of the invention are generally referred to as “fluidicoperations.” For example, where a device's intended function is toscreen a sample against a panel of antigens, the entire screen isreferred to as a single fluidic operation. Similarly, the fluidicoperation of a device intended to amplify nucleic acids is thecompletion of the amplification process, including all of the numerousmelting, annealing extension cycles. However, the individual steps ofthe overall fluidic operation are generally referred to as a “fluidmanipulation.” In the screening example, the combination or mixture of aportion of the sample with a solution containing a single antigen wouldconstitute a fluid manipulation. Similarly, in the amplificationexample, each separate reagent addition step required for each separatecycling step would constitute a single fluid manipulation. In manycases, the fluids utilized in the microfluidic devices and methods ofthe invention are referred to as reactants to denote their ability toundergo a chemical reaction, either alone, or when combined with anotherreactive fluid or composition. It will be readily apparent that thephrases “fluidic operation” and “fluid manipulation” encompass a widevariety of such manipulations for carrying out a variety of chemical,biological and biochemical reactions, either entirely fluid based orincorporating a non-fluid element, e.g., cells, solid supports,catalysts, etc., including, reagent additions, combinations,extractions, filtrations, purifications, and the like.

A “sequencing primer” is an oligonucleotide primer which is can beextended with a polymerase in the presence of a template and appropriatereagents (dNTPs, etc).

DETAILED DESCRIPTION

High throughput manipulation and analysis of fluidic reagents isdesirable for a variety of applications, including nucleic acidsequencing, screening of chemical or biological libraries, purificationof molecules of interest, amplification of nucleic acids and the like.The present invention provides apparatus, systems and methods fordramatically increasing the speed and simplicity of screening,manipulating and assessing fluidic reagents, reagent mixtures, reactionproducts (including the products of DNA sequencing reactions) and thelike. The invention provides integrated systems for performing a varietyof chemical, biochemical and biological experiments and other fluidicoperations, including PCR, DNA sequencing, integrated or sequentialscreening of chemical or biological libraries, and the like. Althoughthe microfluidic systems of the invention are generally described interms of the performance of chemical, biochemical or biologicalreactions separations, incubations and the like, it will be understoodthat, as fluidic systems having general applicability, these systems canhave a wide variety of different uses, e.g., as metering or dispensingsystems in both biological and nonbiological applications.

In the methods of the prior art, most fluidic operations are generallyperformable at the bench scale, e.g., involving reagent volumes rangingfrom 10 μl to 1 or more liters. However, the performance of largenumbers of iterative, successive or parallel fluid manipulations at thebench scale potentially includes a number of associated problems. Forexample, when performed manually, repetitive tasks, e.g., fluidmeasurement and addition, are often plagued by errors and mistakes,which often result in the overall failure of the overall operation.Similarly, iterative or successive processing of small fluid samplesoften results in substantial yield problems, e.g., from loss of materialduring incomplete fluid transfers, i.e., resulting from incompletetransfer of fluid volumes, adsorption of materials on reaction vessels,pipettes and the like. These problems can substantially reduce theaccuracy and reproducibility of a particular process performed manually,or at the bench scale. Further, in fluidic operations that employ largenumbers of parallel fluid manipulations, while the individual separatereactions are not overly cumbersome, the logistics of coordinating andcarrying out each of the parallel manipulations can become unmanageable.Additionally, the costs, complexity and space requirements of equipmentfor facilitating these operations, e.g., robotics, creates furtherdifficulties in performing these types of operations.

In addition to the above, where reagent costs are substantial, even atthe low end of the volume spectrum, a particular fluidic operationinvolving numerous iterative or parallel reagent additions, can becommercially impracticable from a cost standpoint. Further, as reagentvolumes become smaller and smaller, errors in measurement become moreand more problematic. By performing iterative, successive or parallelfluid manipulations in microfluidic devices that are partially sealedand automatable, the above-described problems of measurement and fluidtransfer errors, reagent costs, equipment costs and space requirementsare alleviated.

Accordingly, in one aspect, the present invention provides microfluidicdevices, systems and methods that are particularly useful in performingfluid operations that require a large number of iterative fluidmanipulations. By “iterative fluid manipulations” is meant the movementand/or direction, incubation/reaction, separation or detection ofdiscrete volumes of fluid, typically in a serial format or orientation,in a repetitive fashion, i.e., performing the same type of manipulationon multiple separate samples, diluting a particular sample, etc.,typically while varying one or more parameter in each series ofreactions. When performed at bench scales, iterative fluid manipulationsbecome relatively cumbersome as the number of repetitions becomesgreater, resulting in a substantial increase in the likelihood of errorsin measurement or the like, and requiring massive labor inputs as a userhas to select which parameters or reagents to vary in each successiveoperation. As such, the systems and devices of the present invention areparticularly useful in performing such iterative fluid manipulations,e.g., which require performance of a particular fluid manipulationgreater than about 10 times, typically greater than about 20 times,preferably greater than about 50 times and often greater than about 100times. In particularly preferred aspects, such fluid manipulations arerepeated between about 10 and 100 times or between about 100 and 1000times.

The present invention, therefore, provides microfluidic systems andmethods that are useful for performing a wide variety of differentfluidic operations, i.e., chemical, biochemical or biological reactions,incubations, separations, and the like, which, when performed bypreviously known methods, would be difficult or cumbersome, either interms of time, space, labor and/or costs. In particular, the systems ofthe present invention permit the performance of a wide variety offluidic operations without requiring large amounts of space, expensivereagents and/or equipment, or excessive time and labor costs.Specifically, as microfluidic devices are employed, the methods andsystems of the invention utilize less space and have smaller reagentrequirements. In addition, because these microfluidic systems areautomatable and partially sealed, they can reduce the amount of humaninvolvement in these manipulations, saving labor and eliminating many ofthe areas that are prone to human error, e.g., contamination,measurement errors, loss of materials and the like. A powerful newadditional aspect of the present invention is the ability of theapparatus, systems and methods to select components of iterative assaysbased upon the results of previous assays.

In its simplest embodiment, iterative fluid manipulation includes therepeated movement, direction or delivery of a discrete volume of aparticular reagent to or through a particular reaction chamber orchannel. In more complex embodiments, such iterative fluid manipulationsinclude the apportioning of larger fluid volumes into smaller, discretefluid volumes, which includes the aliquoting of a given sample among anumber of separate reaction chambers or channels, or the taking ofaliquots from numerous discrete fluids, e.g., samples, to deliver thesealiquots to the same or different reaction chambers or channels.

In another, similar aspect, the devices, systems and methods of theinvention are useful in performing fluidic operations that require alarge number of successive fluid manipulations, i.e., in performing anumber of preparative and analytical reactions or operations on a givensample. By “successive fluid manipulations” is generally meant a fluidicoperation that involves the successive treatment of a given fluid samplevolume, i.e., combination/reaction with reactants, incubation,purification/separation, analysis of products, and the like. Wheresuccessive fluid manipulations are performed at the bench scale, e.g.,the performance of numerous, different manipulations on a particularsample such as combination with reagents, incubation, separation anddetection, such manipulations can also become cumbersome as the numberof steps increases, as with each step, the possibility of introducing anerror into the operation or experiment increases. This complexity, andthe consequent increased possibility of errors increases substantiallyas the number of samples to be passed through the operation increases.Thus, the devices or systems of the present invention are alsoparticularly useful in performing fluidic operations which requiresuccessive fluid manipulations of a given sample or fluid of interest,e.g., more than 2 steps or different manipulations, typically greaterthan 5 steps or different manipulations, preferably greater than 10steps or different fluid manipulations. The systems are also useful andreadily capable of performing fluidic operations that include greaterthan 20, 50, 100, 1000 steps or different fluid manipulations on a givenfluid volume.

In a related, but alternate aspect, the devices, systems and methods ofthe invention are useful in performing fluidic operations that require alarge number of parallel fluid manipulations, i.e., to screen biologicalsamples, screen test compounds for drug discovery, e.g., as set forth inU.S. patent application Ser. Nos. 08/671,987 now U.S. Pat. No. 5,779,868and 08/671,986, now U.S. Pat. No. 5,942,493 both filed Jun. 28, 1996 andincorporated herein by reference. To carry out these operations, asubstrate will typically employ an array of parallel channels and/orchannel networks, interconnected by one or more common channels. Fluidsrequired for the subject reaction, e.g., samples or reagents, aredirected along one or more of the common channels, and are delivered toeach of the parallel channels.

As used herein, “parallel fluid manipulations” means the substantiallyconcurrent movement and/or direction, incubation/reaction, separation ordetection of discrete fluid volumes to a plurality of parallel channelsand/or channel networks, or chambers of a microfluidic device, i.e.,greater than about 10 distinct parallel channels or chambers, typicallygreater than 20 distinct channels or chambers, preferably greater thanabout 50 distinct channels or chambers, and often greater than about 100distinct channels or chambers. As used herein, the term “parallel”refers to the ability to concomitantly or substantially concurrentlyprocess two or more separate fluid volumes, and does not necessarilydenote a specific channel or chamber structure or layout.

Ultra high-throughput analysis systems are provided, for example forperforming nucleic acids-based diagnostic and sequencing applications,e.g., in a reference laboratory setting. The system typically hasseveral components: a specimen and reagents handling system; an“operating system” for processing integrated microchip experimentationsteps; application-specific analysis devices (optionally referred inthis application e.g., as “LabChips™” (LabChip™ is a trademark ofCaliper Technologies, Corp., Palo Alto Calif.); a fluorescence-basedsignal detection system, and multiple software components that allow theuser to interact with the system, and run processing steps, interpretdata, and report results.

Application to Sequencing Projects

In a preferred aspect, the invention provides a closed loop device fordetermining the entire sequence of an unknown DNA molecule of interestby iteratively sequencing sub regions of the molecule of interest. Inone aspect, oligonucleotides are chosen from a pool of possiblesequencing primers upon determination of an initial portion of the DNAsequence. With iterative utilization of this strategy, it is possible towalk through an entire sequence without synthesizing new primers.

“Primer walking” is a standard strategy for determining the sequence ofan unknown DNA. For example, a portion of an unsequenced DNA cloned intoa plasmid can be sequenced using a primer complementary to a portion ofthe plasmid, and extending the sequencing reaction into the unknownregion of the DNA with a template dependent polymerase. However,standard electrophoretic analysis of the sequencing reaction only allowsresolution of a few hundred nucleotides. Once the sequence of a fewhundred nucleotides is determined, a second primer is synthesized to becomplementary to a portion of the sequenced region, and the reaction isrepeated, giving a new sequence which yields an additional few hundrednucleotides. Although the process is conceptually simple, it is alsovery labor intensive and time consuming for large nucleotide sequences.For example, sequencing a Yeast Artificial Chromosome (YAC) clone of amodest 100,000 bases using this seral primer walking fashion wouldrequire at least about 300-1,000 individual reactions, with acorresponding number of primer syntheses. It should also be noted thateach of these primer syntheses typically produces thousands of times asmuch primer as needed for the particular sequencing reaction,dramatically increasing the cost of sequencing.

The present invention simplifies the standard primer walking strategy bymodifying, automating and integrating each part of primer walking into asingle integrated system. In the methods of the invention, all of themixing and analysis steps are performed with an integrated system, andall primer synthetic steps are preferably avoided. In brief, a templatenucleic acid is selected and introduced into a reaction channel in amicrofluidic (generally electroosmotic) device of the invention. Thetemplate is optionally amplified, e.g., by introducing PCR or LCRreagents into the channel and performing cycles of heating and coolingon the template. Alternatively, e.g., where the source of template isfrom an abundant sequence such as a cloned nucleic acid, furtheramplification can be unnecessary. In addition to amplificationprocedures, a PCR nuclease chain termination procedure can also be usedfor direct sequencing in the methods of the invention. Porter et al.(1997) Nucleic Acids Research 25(8):1611-1617 describe the biochemistryof PCR chain termination methods.

Sequencing reagents are added to the template nucleic acid and asequencing reaction is performed appropriate to the particular reactionin use. Many appropriate reactions are known, with the Sanger dideoxychain termination method being the most common. See, Sanger et al.(1977) Proc. Nat. Acad. Sci., USA 74:5463-5467. The primer used to primesynthesis is typically selected from a pre-synthesized set of nucleicacid primers, preferably a set including many or all of the primers fora particular primer length. In a preferred aspect, modular primers areused.

After the sequencing reaction is run, the products are separated by sizeand/or charge in an analysis region of the microfluidic device. Asdiscussed herein, the devices of the invention can be used toelectrophoretically separate macromolecules by size and/or charge. Theseparated products are detected, often as they pass a detector (nucleicacids are typically labeled with radioactive nucleotides orfluorophores; accordingly appropriate detectors includespectrophotometers, fluorescent detectors, microscopes (e.g., forfluorescent microscopy) and scintillation counting devices). Detectionof the size separated products is used to compile sequence informationfor the region being sequenced. A computer is used to select a secondprimer from the pre-synthesized primer set which hybridizes to thesequenced region, and the process is iteratively repeated with thesecond primer, leading to sequencing of a second region, selection of athird primer hybridizing to the second region, etc.

Providing DNA Templates for Sequencing

The integrated systems of the invention are useful for sequencing a widevariety of nucleic acid constructs. Essentially any DNA template can besequenced, with the selection of the nucleic acid to be sequenceddepending upon the construct in hand by the sequencer. Thus, an initialstep in the methods of the invention is the selection or production of atemplate nucleic acid to be sequenced.

Many methods of making recombinant ribo and deoxyribo nucleic acids,including recombinant plasmids, recombinant lambda phage, cosmids, yeastartificial chromosomes (YACs), P1 artificial chromosomes, BacterialArtificial Chromosomes (BACs), and the like are known. The sequencing oflarge nucleic acid templates is advantageously performed by the presentmethods, systems and apparatus, because an entire nucleic acid can besequenced by primer walking along the length of the template in severalrapid cycles of sequencing.

Cloning Templates or other Targets for use in the Methods, Apparatus andSystems of the invention

Examples of appropriate cloning techniques for making nucleic acids, andinstructions sufficient to direct persons of skill through most standardcloning and other template preparation exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1997, supplement 37) (Ausubel). Basicprocedures for cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Lewin (1995)Genes V Oxford University Press Inc., NY (Lewin); and Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY.Product information from manufacturers of biological reagents andexperimental equipment also provide information useful in knownbiological methods. Such manufacturers include the Sigma ChemicalCompany (Saint Louis, Mo.); New England Biolabs (Beverly, Mass.); R&Dsystems (Minneapolis, Minn.); Pharmacia LKB Biotechnology (Piscataway,N.J.); CLONTECH Laboratories, Inc. (Palo Alto, Calif.); ChemGenes Corp.,(Waltham Mass.) Aldrich Chemical Company (Milwaukee, Wis.); GlenResearch, Inc. (Sterling, Va.); GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.); Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland); Invitrogen (San Diego, Calif.); Perkin Ehner(Foster City, Calif.); and Strategene; as well as many other commercialsources known to one of skill.

In one aspect, the generation of large nucleic acids is useful inpracticing the invention. It will be appreciated that such templates areparticularly useful in some aspects where the methods and devices of theinvention are used to sequence large regions of DNA, e.g., for genomicstypes of applications. An introduction to large clones such as YACs,BACs, PACs and MACs as artificial chromosomes is provided by Monaco andLarin (1994) Trends Biotechnol 12 (7): 280-286.

The construction of nucleic acid libraries of template nucleic acids isdescribed in the above references. YACs and YAC libraries are furtherdescribed in Burke et al. (1987) Science 236:806-812. Gridded librariesof YACs are described in Anand et al. (1989) Nucleic Acids Res. 17,3425-3433, and Anand et al. (1990) Nucleic Acids Res. Riley (1990)18:1951-1956 Nucleic Acids Res. 18(10): 2887-2890 and the referencestherein describe cloning of YACs and the use of vectorettes inconjunction with YACs. See also, Ausubel, chapter 13. Cosmid cloning isalso well known. See, e.g., Ausubel, chapter 1.10.11 (supplement 13) andthe references therein. See also, Ish-Horowitz and Burke (1981) NucleicAcids Res. 9:2989-2998; Murray (1983) Phage Lambda and Molecular Cloningin Lambda II (Hendrix et al., eds) 395-432 Cold Spring HarborLaboratory, NY; Frischauf et al. (1983) J.Mol. Biol. 170:827-842; and,Dunn and Blattner (1987) Nucleic Acids Res. 15:2677-2698, and thereferences cited therein. Construction of BAC and P1 libraries is wellknown; see, e.g., Ashworth et al. (1995) Anal Biochem 224 (2): 564-571;Wang et al. (1994) Genomics 24(3): 527-534; Kim et al. (1994) Genomics22(2): 336-9; Rouquier et al. (1994) Anal Biochem 217(2): 205-9; Shizuyaet al. (1992) Proc Natl Acad Sci U S A 89(18): 8794-7; Kim et al. (1994)Genomics 22 (2): 336-9; Woo et al. (1994) Nucleic Acids Res 22(23):4922-31; Wang et al. (1995) Plant (3): 525-33; Cai (1995) Genomics 29(2): 413-25; Schmitt et al. (1996) Genomics 1996 33(1): 9-20; Kim et al.(1996) Genomics 34(2): 213-8; Kim et al. (1996) Proc Natl Acad Sci U S A(13): 6297-301; Pusch et al. (1996) Gene 183(1-2): 29-33; and, Wang etal. (1996) Genome Res 6(7): 612-9. In general, where the desired goal ofa sequencing project is the sequencing of a genome or expression profileof an organism, a library of the organism's cDNA or genomic DNA is madeaccording to standard procedures described, e.g., in the referencesabove. Individual clones are isolated and sequenced, and overlappingsequence information is ordered to provide the sequence of the organism.See also, Tomb et al. (1997) Nature 539-547 describing the whole genomerandom sequencing and assembly of the complete genomic sequence ofHelicobacter pylori; Fleischmann et al. (1995) Science 269:496-512describing whole genome random sequencing and assembly of the completeHaemophilus influenzae genome; Fraser et al. (1995) Science 270:397-403describing whole genome random sequencing and assembly of the completeMycoplasma genitalium genome and Bult et al. (1996) Science273:1058-1073 describing whole genome random sequencing and assembly ofthe complete Methanococcus jannaschii genome.

The nucleic acids sequenced by this invention, whether RNA, cDNA,genomic DNA, or a hybrid of the various combinations, are isolated frombiological sources or synthesized in vitro. The nucleic acids of theinvention are present in transformed or transfected whole cells, intransformed or transfected cell lysates, or in a partially purified orsubstantially pure form.

Amplification in Microscale Devices—PCR

Bench scale in vitro amplification techniques suitable for amplifyingsequences to provide a nucleic acid e.g., as a diagnostic indicator forthe presence of the sequence, or for subsequent analysis, sequencing orsubloning are known.

In brief, the most common form of in vitro amplification, i.e., PCRamplification, generally involves the use of one strand of the targetnucleic acid sequence as a template for producing a large number ofcomplements to that sequence. As used herein, the phrase “target nucleicacid sequence” generally refers to a nucleic acid sequence, or portionof a nucleic acid sequence that is the subject of a particular fluidicoperation, e.g., analysis, amplification, identification or the like.Generally, two primer sequences complementary to different ends of asegment of the complementary strands of the target sequence hybridizewith their respective strands of the target sequence, and in thepresence of polymerase enzymes and nucleoside triphosphates, the primersare extended along the target sequence through the action of thepolymerase enzyme. The extensions are melted from the target sequence byraising the temperature of the reaction mixture, and the process isrepeated, this time with the additional copies of the target sequencesynthesized in the preceding steps. PCR amplification typically involvesrepeated cycles of denaturation, hybridization and extension reactionsto produce sufficient amounts of the target nucleic acid, all of whichare carried out at different temperatures. Typically, melting of thestrands, or heat denaturation, involves temperatures ranging from about90° C. to 100° C. for times ranging from seconds to minutes. Thetemperature is then cycled down, e.g., to between about 40° C. and 65°C. for annealing, and then cycled up to between about 70° C. and 85° C.for extension of the primers along the target strand.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques (e.g., NASBA) are found inBerger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S.Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis);Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIHResearch (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874;Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988)Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wuand Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117,and Sooknanan and Malek (1995) Biotechnology 13: 563-564. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace et al., U.S. Pat. No. 5,426,039. Improved methods of amplifyinglarge nucleic acids by PCR are summarized in Cheng et al. (1994) Nature369: 684-685 and the references therein, in which PCR amplicons of up to40 kb are generated. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausbel, Sambrook and Berger, all supra.

It will be appreciated that these benchtop uses for PCR are adaptable tomicrofluidic systems. Indeed, PCR amplification is particularly wellsuited to use in the apparatus, methods and systems of the invention.

Thermocycling amplification methods, including PCR and LCR, areconveniently performed in microscale devices, making iterative fluidicoperations involving PCR well suited to use in methods and devices ofthe present invention (see also, U.S. Pat. Nos. 5,498,392 and 5,587,128to Willingham et al.). Accordingly, in one preferred embodiment,generation of amplicons such as sequencing templates using PCR, ordirect sequencing of nucleic acids by PCR (e.g., using nucleasedigestion as described supra) is performed with the integrated systemsand devices of the invention.

Thermocycling in microscale devices is described in co-pendingapplication U.S. Ser. No. 60/056058, entitled “ELECTRICAL CURRENT FORCONTROLLING FLUID TEMPERATURES IN MICROCHANNELS” filed Sep. 2, 1997 byCalvin Chow, Anne R. Kopf-Sill and J. Wallace Parce and in Ser. No.08/977,528, U.S. Pat. No. 5,965,410 filed Nov. 25, 1997. In brief,energy is provided to heat fluids, e.g., samples, analytes, buffers andreagents, in desired locations of the substrates in an efficient mannerby application of electric current to fluids in microchannels. Thus, thepresent invention optionally uses power sources that pass electricalcurrent through the fluid in a channel for heating purposes, as well asfor material transport. In exemplary embodiments, the fluid passesthrough a channel of a desired cross-section (e.g., diameter) to enhancethermal transfer of energy from the current to the fluid. The channelscan be formed on almost any type of substrate material such as, forexample, amorphous materials (e.g., glass, plastic, silicon),composites, multi-layered materials, combinations thereof, and the like.

In general, electric current passing through the fluid in a channelproduces heat by dissipating energy through the electrical resistance ofthe fluid. Power dissipates as the current passes through the fluid andgoes into the fluid as energy as a function of time to heat the fluid.The following mathematical expression generally describes a relationshipbetween power, electrical current, and fluid resistance:

POWER=I²R

where

POWER=power dissipated in fluid;

I=electric current passing through fluid; and

R=electric resistance offluid.

The above equation provides a relationship between power dissipated(“POWER”) to current (“I”) and resistance (“R”). In some of theembodiments, which are directed toward moving fluid in channels, e.g.,to provide mixing, electrophoretic separation, or the like, a portion ofthe power goes into kinetic energy of moving the fluid through thechannel. However, it is also possible to use a selected portion of thepower to controllably heat fluid in a channel or selected channelregions. A channel region suitable for heating is often narrower orsmaller in cross-section than other channel regions in the channelstructure, as a smaller cross-section provides higher resistance in thefluid, which increases the temperature of the fluid as electric currentpasses through. Alternatively, the electric current is increased acrossthe length of the channel by increased voltage, which also increases theamount of power dissipated into the fluid to correspondingly increasefluid temperature.

To selectively control the temperature of fluid at a region of thechannel, a power supply applies voltage and/or current in one of manyways. For instance, a power supply can apply direct current (i.e., DC)or alternating current (AC), which passes through the channel and into achannel region which is smaller in cross-section, thereby heating fluidin the region. This current is selectively adjusted in magnitude tocomplement any voltage or electric field that is applied to move fluidin and out of the region. AC current, voltage, and/or frequency can beadjusted, for example, to heat the fluid without substantially movingthe fluid. Alternatively, a power supply can apply a pulse or impulse ofcurrent and/or voltage, which passes through the channel and into achannel region to heat fluid in the region. This pulse is selectivelyadjusted to complement any voltage or electric field that is applied tomove fluid in and out of the region. Pulse width, shape, and/orintensity can be adjusted, for example, to heat the fluid substantiallywithout moving the fluid or to heat the fluid while moving the fluid.Still further, the power supply can apply any combination of DC, AC, andpulse, depending upon the application. In practice, direct applicationof electric current to fluids in the microchannels of the inventionresults in extremely rapid and easily controlled changes in temperature.

A controller or computer such as a personal computer monitors thetemperature of the fluid in the region of the channel where the fluid isheated. The controller or computer receives current and voltageinformation from, for example, the power supply and identifies ordetects temperature of fluid in the region of the channel. Dependingupon the desired temperature of fluid in the region, the controller orcomputer adjusts voltage and/or current to meet the desired fluidtemperature. The controller or computer also can be set to be “currentcontrolled” or “voltage controlled” or “power controlled” depending uponthe application.

The region which is heated can be a “coil” which is optionally in aplanar arrangement. Transfer of heat from the coil to a reaction channelthrough a substrate material is used to heat the reaction channel.Alternatively, the coil itself is optionally the reaction channel.

A voltage is applied between regions of the coil to direct currentthrough the fluid for heating purposes. In particular, a power supplyprovides a voltage differential between regions of the coil. Currentflows between the regions and traverses a plurality of coils or coilloops (which can be planar), which are defined by a substrate. Shape andsize of the coils can influence an ability of current to heat the fluidin the coil. As current traverses through the fluid, energy istransferred to the fluid for heating purposes. Cooling coils can also beused. As a cooling coil, a fluid traverses from region to region in thecoil, which can be placed to permit heat transfer through a substratefrom a sample. The cooling fluid can be a variety of substancesincluding liquids and gases. As merely an example, the cooling fluidincludes aqueous solutions, liquid or gaseous nitrogen, and others. Thecooling fluid can be moved between regions using any of the techniquesdescribed herein, and others. Further details are found in Chow et al.,supra.

The introduction of electrical current into fluid causes heat (Jouleheating). In the examples of fluid movement herein where thermal effectsare not desired, the heating effect is minimal because, at the smallcurrents employed, heat is rapidly dissipated into the chip itself. Bysubstantially increasing the current across the channel, rapidtemperature changes are induced that can be monitored by conductivity.At the same time, the fluid can be kept static in the channel by usingalternating instead of direct current. Because nanoliter volumes offluid have tiny thermal mass, transitions between temperatures can beextremely short. Oscillations between any two temperatures above 0° C.and below 100° C. in 100 milliseconds have been performed.

Joule heating in microchannels is an example of how a key component of aconventional genomics methods can be dramatically improved in theformats provided herein. PCR takes hours to perform currently, primarilybecause it takes a long time for conventional heating blocks tooscillate between temperatures. In addition, reagent cost is an obstacleto massive experimentation. Both these parameters are altered by ordersof magnitude in the LabChip format. FIG. 1 shows amplification ofbacteriophage lambda DNA in a 10 nanoliter volume. It should be notedthat the optical interrogation volume was 400 picoliters. At a templateconcentration of 10 ng/ml, the signal seen starting at the 27th cyclecame from the amplification of approximately 80 target molecules. Thetransition between 68° C. and 94° C. took place in less than 1 second.

In one aspect, PCR reaction conditions are controlled as a function ofchannel geometry. Microfabrication methods permit the manufacture ofchannels that have precise variations in cross sectional area. Since thechannel resistance is inversely proportional to the cross sectionalarea, the temperature varies with the width and depth of the channel fora given flow of current. As fluid moves through a structure of varyingcross sectional area, its temperature will change, depending on thedimensions of the channel at any given point. The amount of time itexperiences a given temperature will be determined by the velocity ofthe fluid flow, and the length of channel with those dimensions. Thisconcept is illustrated in FIG. 2. Nucleic acids of typical lengths havea low diffusion efficient (about 10⁻⁷ cm/sec²). Thus over the time framenecessary to affect amplification, DNA will only diffuse a few hundredmicrons. In a given channel, reactions of a few nanoliters will occupy afew millimeters. Thus in devices of convenient length (a fewcentimeters), many PCR reactions can be performed concurrently yieldingnew amplification products every few seconds per channel. In parallelformats, hundreds of separate reactions can be performed simultaneously.Because of its simplicity, throughput and convenience, thisamplification unit is a preferred feature of many assays herein.

In FIG. 2, amplification reactions are performed concurrently in seriesusing biased alternating current to heat the fluid inside the channeland move material through it. The time for each step of the reaction iscontrolled by determining the speed of movement and the length ofchannel having particular widths. Flow can be reversed to allow a singlesmall channel region to be used for many separate amplifications.

As depicted, several samples are run simultaneously in channel 210.Sample 215 is in narrow channel region 220; in operation, this region isheated to, e.g., 95° C. (hot enough to denature nucleic acids present insample 215, but cool enough that thermostable reagents such as Taq DNApolymerase are relatively stable due to the relative size of region 220and the applied current. Concurrently, wide channel region 230 isheated, e.g., to 60° C. (cool enough for binding of primers in sample225 and initiation of polymerase extension), due to the relative size ofregion 230 and the applied current. Concurrently, intermediate channelregion 235 is heated, e.g., to 72° C. (hot enough to prevent unwantednon-specific primer-target nucleic acid interactions in sample 240 andcool enough to permit continued polymerase extension), due to therelative size of region 235 and the applied current. This process can beconcurrently carried out with a plurality of additional channel regionssuch as narrow region 245, wide region 250 and intermediate region 255,with samples 260, 265 and 270.

Where possible, direct detection of amplified products can be employed.For example, differentially labeled competitive probe hybridization isused for single nucleotide discrimination. Alternatively, molecularbeacons or single nucleotide polymerase extension can be employed.Homogeneous detection by fluorescence polarization spectroscopy can alsobe utilized (fluorescence polarization has been used to distinguishbetween labeled small molecules free in solution or bound to proteinreceptors).

If the analysis requires post-PCR processing, a more complex channel andcontrol structure is used as in the case where the amplified product isto be typed at a microsatellite locus. Because single nucleotideseparations take time (approximately 5 minutes today), the output of theserial amplification unit is optionally analyzed in parallel separationschannels following serial to parallel fluidic manipulation as describedherein.

Where possible, direct detection of amplified products can be employed.For example, differentially labeled competitive probe hybridization isused for single nucleotide discrimination. Alternatively, molecularbeacons or single nucleotide polymerase extension can be employed.Homogeneous detection by fluorescence polarization spectroscopy can alsobe utilized (fluorescence polarization has been used to distinguishbetween labeled small molecules free in solution or bound to proteinreceptors).

Amplification in Microscal Devices—Non-thermal Methods

Another example of a fluidic operation requiring multiple iterativefluid manipulations which was previously impracticable for cost reasons,is non-thermal amplification of nucleic acids. In non-thermalamplification, strand separation is optionally carried out by chemicalmeans. Thus, by “non-thermal amplification” is meant the amplificationof nucleic acids without thermal cycling of the reaction mixture toaffect the melting and annealing of the nucleic acid strands. Inpractice, such methods involve the chemical denaturation of nucleic acidstrands, followed by dilution or neutralization of the chemicaldenaturant. For example, in one aspect, strand separation is carried outby raising the pH of the reaction mixture to denature the nucleic acidstrands. The pH is then returned to neutral, for annealing andextension. Other chemical denaturants are equally useful to affectstrand separation. For example, chaotropic agents, e.g., urea,formamide, and the like, are employed in place of base.

Regardless of the chemical denaturant, however, addition of thesematerials will typically result in the denaturing of the enzymes presentin the reaction mixture, e.g., polymerases, in addition to the nucleicacids, and thereby lead to their inactivation. As such, performance ofthis type of amplification at the bench scale, would require largeamounts of expensive enzymes. Further, the additional volume requiredfor adding these enzymes, as well as diluting or neutralizing thedenaturants, would result in cumbersome manipulations, particularlywhere a large number of cycles is performed.

In the systems, devices and methods of the present invention,non-thermal amplification can be carried out by introducing a sample ortarget nucleic acid into a reaction chamber, channel or zone of amicrofluidic device. The complementary strands of the target nucleicacid are melted apart by introducing a preselected volume of a chemicaldenaturant, which denatures the complementary strands of the nucleicacid. In particularly preferred aspects, denaturation is accomplished byraising the pH of the reaction mixture to approximately 10-13. This isreadily accomplished by introducing an equal volume of dilute NaOH,e.g., approximately 0.2N NaOH).

Annealing of the primers to the target strand is carried out by removingthe denaturing effects of the denaturant. For example, in those aspectswhere a dilute base is used to denature the nucleic acid, the base isoptionally neutralized by the addition of a similar volume of diluteacid, e.g., 0.2N HCl. Where chaotropic agents are used, the denaturingeffect can generally be removed by desalting the reaction mixture or thelike. A preselected volume containing an effective amount of polymeraseenzyme and primer sequences are then added to the reaction mixture,i.e., sufficient to amplify the target sequence. Because volumes ofreagents are so small in the devices and methods of the invention, thepolymerase need not be thermally or otherwise stable to the more extremeconditions of the amplification reaction as in PCR. Specifically,denaturation of the nucleic acids will typically result in denaturationof the polymerase enzyme, as well. However, additional amounts of enzymecan be added back to the amplification mixture. Because small volumesare used, the costs are maintained relatively low. As a result of this,any number of a variety of common polymerase enzymes can be used,including E. coli DNA polymerases, e.g., E. coli DNA pol I, Klenowfragment, T7 DNA polymerase or the like. Further, one could operate thesystem at an elevated temperature and utilize thermally stable Taqpolymerases, Pfu DNA polymerase, Bst and Vent, all of which arecommercially available.

The primers anneal to the target nucleic acid and begin the extensionprocess. Denaturation, annealing and extension steps are then repeatedthe desired number of times to sufficiently amplify the target nucleicacid. Typically, these cycles are repeated from about 10 to about 100times, and preferably between about 10 and 50 times.

A number of modifications are readily made to this amplificationprocess. For example, one can introduce primer sequences into thereaction mixture at the outset, or along with the polymerase enzymes, asindicated. Similarly, following denaturation, it can be desirable todesalt the amplification reaction mixture, e.g., by passing the mixturethrough a chromatographic matrix incorporated into the device or byseparating the desired elements of the reaction mixture byelectrophoresing the mixture in an appropriate medium. Such desaltingcan be particularly useful where other chemical denaturants are used,e.g., urea, etc. In such cases, the denaturing effects of thesechemicals are typically removed by dilution or removal of the denaturantfrom the amplification reaction mixture, i.e., by desalting.

An example of a microfluidic device for practicing non-thermalamplification is illustrated in FIG. 3. For ease of discussion, theoperation of this device is described with reference to the use of base(NaOH) mediated denaturation and neutralization with acid (HCl). Asshown, the device 300 is illustrated as being fabricated in a planarsubstrate 301, and including a main channel 302 originating from samplereservoir 304 and terminating in waste reservoir 306. The device alsoincludes a transverse channel 308 which intersects the main channel, andhas at its termini, buffer reservoir 310 and waste reservoir 312. Mainchannel 302 is alternately intersected by NaOH introduction channels(314 a, 314 b and 314 c) fluidly connected to reservoirs which containan appropriate concentration of NaOH (316 a, 316 b and 316 c,respectively) and HCl introduction channels (318 a, 318 b and 318 c)which are fluidly connected to reservoirs (320 a, 320 b and 320 c,respectively) which contain an appropriate concentration of HCl, forneutralizing the base. In the direction of flow along the main channel302, from the sample reservoir 304 to the waste reservoir 306, aftereach intersection of the main channel 302 with the HCl introductionchannels, 318 a, 318 b and 318 c, there is disposed within the mainchannel, a desalting region 322 a, 322 b and 322 c. e.g., a portion ofthe channel that includes an appropriate gel exclusion matrix, nucleicacid binding region, or the like, for separating the salts present inthe sample fluid from the amplified nucleic acid. After the desaltingregions, the main channel is intersected by enzyme/NTP introductionchannels 324 a, 324 b and 324 c, which are fluidly connected toreservoirs (326 a, 326 b and 326 c) which contain effective amounts ofan appropriate DNA polymerase, as well as the four nucleosidetriphosphates or deoxynucleoside triphosphates (NTPs). A detectionwindow 328 is shown across the main channel 302 near the terminus of thechannel into waste reservoir, to detect the product of the overallamplification process. Optional separation regions are also provided inthe terminal portion of the main channel 302 between the last desaltingregion 322 c and the final waste reservoir 306.

In operation, a sample containing a nucleic acid of interest, e.g., thatis sought to be amplified, is introduced along with appropriate primersequences into main channel 302, e.g., via sample reservoir 304. Astream of sample/primer is transported along main channel 302 and out towaste reservoir 312 along transverse channel 308, e.g., by applyingappropriate voltages at the various reservoirs, as described herein. Ameasured slug of sample/primer is then pumped into main channel 302.Slugs of sample are optionally introduced from an external source, e.g.,from a sampling system, e.g., as described in commonly assignedcopending U.S. patent application Ser. No. 08/671,986 filed Jun. 28,1996, and U.S. patent application Ser. No. 08/760,446, filed Dec. 6,1996, each of which is incorporated herein by reference in its entiretyfor all purposes.

Following introduction into the device, the sample/primer mixture isthen transported up to the intersection of main channel 302 and baseintroduction channel 314 a, whereupon the sample is mixed with a streamof NaOH, that is delivered into main channel 302 from reservoir 316 a,thereby denaturing the nucleic acid of interest. The denaturedsample/primer mixture continues down main channel until it reaches theintersection of the main channel with the HCl introduction channel 318a, whereupon the denatured sample/primer mixture is mixed with the HCl,thereby neutralizing the mixture and allowing the denatured strands tore-anneal with the primer sequences.

Following this annealing step, the annealed mixture is then transportedthrough a desalting region 322 a, to separate the nucleic acid/primersof interest from salts and low molecular weight contaminants. Thedesalted, annealed mixture then continues down the main channel until itreaches the intersection of the main channel 302 with enzyme/NTPintroduction channel 324 a, whereupon the mixture is mixed with aneffective amount of DNA polymerase enzyme in combination with effectiveamounts of the four NTPs used for amplification, and other requisitecomponents for amplification, e.g., Mg++, KCl, etc., whereupon theenzyme will catalyze extension of the primers along the template nucleicacid of interest.

This process of denaturing/annealing and extending the nucleic acid ofinterest is continued along the main channel for the desired number ofcycles. Although the illustrated device only shows sufficientdenaturant/neutralizer/enzyme channels for three cycles, this is solelyfor ease of discussion. It will be readily appreciated that the numberof cycles can be readily increased by increasing the number of suchchannels in the device.

It will be readily apparent that a number of different channelgeometries er effective in producing the non-thermal amplificationdevices and systems of the present invention.

Synthesis and Selection of Primers and Primer Sets—Application toMicrofluidic Sequencing

Oligonucleotides for use as primers or probes, e.g., in sequencing orPCR or non-thermal amplification reactions in microfluidic apparatus aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981), Tetrahedron Letts., 22(20): 1859-1862, e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al. (1984) NucleicAcids Res., 12:6159-6168. Oligonucleotides can also be custom made andordered from a variety of commercial sources known to persons of skill.Purification of oligonucleotides, where necessary, is typicallyperformed by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom.255:137-149. The sequence of the synthetic oligonucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560.

While primers can hybridize to any of a number of sequences, selectingoptimal primers is typically done using computer assisted considerationof available sequences and excluding potential primers which do not havedesired hybridization characteristics, and/or including potentialprimers which meet selected hybridization characteristics. This is doneby determining all possible nucleic acid primers, or a subset of allpossible primers with selected hybridization properties (e.g., thosewith a selected length, G:C ratio, uniqueness in the given sequence,etc.) based upon the known sequence. The selection of the hybridizationproperties of the primer is dependent on the desired hybridization anddiscrimination properties of the primer. In general, the longer theprimer, the higher the melting temperature. In addition, it is moredifficult to generate a set of primers which includes all possibleoligonucleotides for a given length, as the required number of primersincreases exponentially. For example, all possible 3-mers requires 4³primers, all possible 4-mers requires 4⁴ primers, all possible 5-mersrequires 4⁵ primers, all possible 6-mers requires 4⁶ primers, etc.Standard sequencing primers are often in the range of 15-20 nucleotidesin length, which would require sets of 4¹⁵ to 4²⁰ nucleotides, or1.1×10⁹ to 1.1×10¹² primers.

While it is possible to make such large sets of primers usingcombinatorial chemical techniques, the associated problems of storingand retrieving billions or even trillions of primers make these primersets less desirable. Instead, smaller sets of primers used in a modularfashion are desirable.

For example, Ulanovsky and co-workers have described the mechanism ofthe modular primer effect (Beskin et al. (1995) Nucleic Acids Research23(15):2881-2885) in which short primers of 5-6 nucleotides canspecifically prime a template-dependent polymerase enzyme when 2-3contiguously annealing, but unligated, primers are used in a polymerasedependent reaction such as a sequencing reaction. Polymerase enzymes arepreferentially engaged by longer primers, whether modular orconventional, accounting for the increased specificity of modularprimers. Because it is possible to synthesize easily all possibleprimers with 5-6 nucleotides (i.e., 4⁵ to 46 or 1024 to 4096 primers),it is possible to generate and utilize a universal set of nucleotideprimers, thereby eliminating the need to synthesize particular primersto extend nucleotide sequencing reactions of nucleotide templates. In analternative embodiment, a ligase enzyme is used to ligate primers whichhybridize to adjacent portions of a template, thereby providing a longerprimer.

A modified version of the use of the modular primer strategy, in whichsmall nucleotide primers are specifically elongated for use in PCR toamplify and sequence template nucleic acids has also been described. Theprocedure is referred to as DNA sequencing using differential extensionwith nucleotide subsets (DENS). See, Raja et al. (1997) Nucleic AcidsResearch 25(4):800-805. Thus, whether standard Sanger-style sequencingor direct PCR sequencing using boronated nucleotides and a nuclease(see, Porter et al. 1997, supra.) are desired, small sets of shortprimers are sufficient for use in sequencing and PCR and are desirable.

It is expected that one of skill is thoroughly familiar with the theoryand practice of nucleic acid hybridization and primer selection. Gait,ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford(1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K.L. Dueholm J.. Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methodsin Molecular Biology, volume 20; and Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology—hybridization withnucleic acid probes, e.g., part I chapter 2 “overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y. provide a basic guide to nucleic acid hybridization. Innis supraprovides an overview of primer selection.

One of skill will recognize that the 3′ end of an amplification primeris more important for PCR than the 5′ end. Investigators have reportedPCR products where only a few nucleotides at the 3′ end of anamplification primer were complementary to a DNA to be amplified. Inthis regard, nucleotides at the 5′ end of a primer can incorporatestructural features unrelated to the target nucleic acid; for instance,in one embodiment, a sequencing primer hybridization site (or acomplement to such as primer, depending on the application) isincorporated into the amplification primer, where the sequencing primeris derived from a primer used in a standard sequencing kit, such as oneusing a biotinylated or dye-labeled universal M13 or SP6 primer. Thesestructural features are referred to as constant primer regions. Theprimers are typically selected so that there is no complementaritybetween any known target sequence and any constant primer region. One ofskill will appreciate that constant regions in primer sequences areoptional.

The primers are selected so that no secondary structure forms within theprimer. Self-complementary primers have poor hybridization properties,because the complementary portions of the primers self hybridize (i.e.,form hairpin structures). Modular primers are selected to have minimalcross-hybridization, thereby preventing competition between individualprimers and a template nucleic acid and preventing duplex formation ofthe primers in solution, and possible concatenation of the primersduring PCR. If there is more than one constant region in the primer, theconstant regions of the primer are selected so that they do notself-hybridize or form hairpin structures.

One of skill will recognize that there are a variety of possible ways ofperforming the above selection steps, and that variations on the stepsare appropriate. Most typically, selection steps are performed usingsimple computer programs to perform the selection as outlined above;however, all of the steps are optionally performed manually. Oneavailable computer program for primer selection is the MacVector™program from Kodak. An alternate program is the MFOLD program (GeneticsComputer Group, Madison Wis.) which predicts secondary structure of,e.g., single-stranded nucleic acids. In addition to programs for primerselection, one of skill can easily design simple programs for any or allof the preferred selection steps.

Alternative Sequencing Strategies

Although the present invention is described for exemplary purposes asusing enzymatic sequencing methods (e.g., using the chain terminationmethods of Sanger, or the exonuclease/PCR methods described above), itwill be appreciated that sequencing by hybridization protocols andchemical degradation protocols are also adapted to use in the presentinvention.

In chemical degradation methods, the template is typically end-labeledwith a radio-active or florescent label and then degraded using thewell-known Maxam-Gilbert method. As applied to the present invention,the chemicals used to degrade the nucleic acid are sequentiallycontacted to the template and the resulting size fragments detected byelectrophoresing the fragments through a microchannel as describedsupra.

Sequencing by hybridization is generally described, e.g., in U.S. Pat.No. 5,202,231, to Drmanac et al. and, e.g., in Drmanac et al. (1989)Genomics 4:114-128. As adapted to the present invention, a microfluidicdevice is provided having a source of labeled primers as describedherein, and a source of template to be sequenced. The template and thelabeled primer are hybridized under highly stringent conditions, whichpermit hybridization to occur only if the primers are perfectlycomplementary to the template. In one embodiment, primers havingcomplementarity to a known region and also having an additional base oradditional bases at the 3′ or 5′ end are separately hybridized to thetemplate; those primers which are perfectly complementary to thetemplate (i.e., where the known and additional bases are perfectlycomplementary to the template) are detected. From the detection of theadditional base or bases, additional primers are selected and theprocess is repeated. Using this strategy, it is possible to sequence theentire template nucleic acid.

Typically, the sequence is extended by only a single base with eachspecific hybridization. This is because, as described supra, it iseasier to make complete sets of small oligonucleotides (e.g., there areonly 4,096 6 nucleotide primers) than it is to make complete sets oflarge oligonucleotides. However, several bases are optionally detectedusing larger primers. One advantage of detecting larger regions ofcomplementarity is that, on average, it is more efficient. It will beappreciated that it is not necessary to test all possible sequences forspecific hybridization if more bases than one adjacent to the knownregions are present in the primers used in the sequencing byhybridization reaction. This is because bases are tested sequentiallyonly until a perfectly complementary sequence to the template is found.Once this primer is determined, additional possible primers for thisregion are not tested; instead, the process is repeated to detect theflanking region. Commonly, the primers have between about 1 and about 4nucleotides which flank known regions of complementarity.

The detection of hybridization is carried out as described supra.Typically, the template is captured in a region of the microfluidicsubstrate and primers are sequentially contacted to the capturedtemplate under stringent hybridization conditions. After hybridizationand detection of hybridization (e.g., by tracking a fluorescent or aradio-active label on the primer) the primer is washed off of thetemplate (e.g., by varying the salt concentration or heat at the site ofhybridization) and the process is repeated with a second primer.

Integrated Fluidic Operations

In addition to sequencing applications, the microfluidic devices andmethods herein are useful in performing other operations that rely upona large number of iterative individual fluid manipulations, e.g.,reagent additions, combinations, apportionings, etc.

Serial Diluter

The simplest illustration of iterative fluid manipulations in amicrofluidic system is in the serial dilution of fluids. Dilution ofsamples, reagents and the like, is a particularly problematic area inmicrofluidic devices. In particular, when operating at extremely smallvolumes, bleed over effects, diffusion and the like prevent the accuratecontrol and transport of fluids, thereby effectively limiting thedynamic range of dilution available through the device. Accordingly, oneachieves a greater dynamic range of dilution by performing iterativeserial dilutions of a sample fluid. In particular, rather than making asingle 1:100 or 1:1000 dilution, one serially makes 1:10 dilutions toachieve the desired dilution. Because each dilution is relatively minor,fluid control is not as substantial a problem.

In the devices of the present invention, dilution of a sample istypically carried; a device that includes a main channel intersected byone or more diluent channels, which are in fluid communication with oneor more diluent reservoirs, respectively. A sample or reagent istransported, e.g., electroosmotically, along the main channel. Diluentis then transported into the main channel and allowed to mix with thesample, reagent or other fluid for which dilution is sought. Control ofthe relative volumes of sample and diluent is affected by controllingthe electrical fields applied to each of these solutions and which drivetheir electroosmotic flow within the system, as described above. Byincorporating multiple diluent channels, one can further increase therange of dilution of which the device is capable.

Integrated Systems for Assay Normalization

One similar application of the integrated systems of the invention isthe titration of assay components into the dynamic range of an assay.For example, an assay can first be performed where one or morecomponents of the assay are not within the a range necessary foradequate performance of the assay, e.g., if the assay is performed usinga concentration which is too high or too low for some components, theassay may not provide quantitative results. This need to titrate assaycomponents into the dynamic range of an assay typically occurs where oneor more component of the assay is present at an unknown activity orconcentration. Ordinarily, the assay must be run at severalconcentrations of components, i.e., the assay is run a first time,components are diluted, the assay is run a second time, etc. until theassay can be performed within the dynamic range of the assay. It will beappreciated that this iterative approach can involve several unknownconcentrations simultaneously, requiring considerable trial and error.

In the integrated systems of the invention, an assay can be performed atas many concentrations of components as necessary to titrate the assaycomponents into the dynamic range of the assay, with the results of eachassay being used to optimize additional assay points. Similarly,titration curves, which are often the result of multiple assay runs withdifferent component concentrations are determined by performing repeatedassays with different concentrations of components. Differentconcentrations of assay components in separate assays can be monitoredserially or in parallel.

The ability to titrate and optimize assays is useful for diagnosticassays, for determining concentrations or activities of selectedcomponents in a system (proteins, enzymes, nucleic acids, smallmolecules, etc.). Furthermore, the present integrated systems providefor rational selection of assay conditions as data is acquired. Forexample, in one embodiment, a diagnostic assay needs to be performedusing several components which are present at initially unknownconcentrations or activities. A first series of concentration oractivity assays is performed to determine the activity or concentrationof particular components, e.g., enzyme, protein, inhibitor, co-factor,nucleic acid, or the like. After these assays are performed and theconcentrations or activities of some or all of the components for thediagnostic assay are determined, the integrated system selectsappropriate amounts of the assay components, performs any necessarydilutions, combines the assay components and performs the diagnosticassay. Similarly, further data points can be collected by adjusting theconcentrations or amounts of diagnostic assay components and re-runningthe assay. All of the fluid manipulations are performed rapidly and theintegrated system is able to assess and compile the results ofindividual data points or individual assays to select which additionalassays need to be performed for assay verification.

In its most basic form, assay optimization involves the identificationof all factors affecting a reaction result, followed by the systematicvariation of each of these variables until optimal reaction conditionsare identified. This is generally termed an “OFAT” method for “onefactor at a time.” Thus, assuming a simple two reagent reaction, onewould first identify the factors affecting the outcome, e.g.,concentration of reagent A, concentration of reagent B and temperature.One would then run the assay where one factor was varied while theothers remained constant. For example, one would perform the samereaction at numerous different concentrations of reagent A, whilemaintaining the concentration of reagent B and the temperature. Next,reagent B would be varied while reagent A and temperature remainedconstant, and finally, the temperature would be varied.

Even in this simplest form, the number and complexity of necessaryreactions is apparent. When one considers that most reactions will havefar more than three variables, and that these variable will not beindependent of each other, the possibility of manually performing theseassays, or even performing them in currently available automated formatsbecomes a daunting prospect. For example, while robotic systems usingmicrowell plates can perform large numbers of manipulations to optimizeassay parameters, such systems are very expensive. Further, as thesesystems are typically limited to the bench scale volumes describedabove, they require large volumes of reagents and large amounts of spacein which to operate.

The devices, systems and methods of the present invention permit theoptimization of large numbers of different assays, by providing anextremely low volume, automatable and sealed format in which suchoptimization can occur rapidly and automatically. For example, thedevices can run a first fluidic operation by combining a preselectedvolume of a first reactant with a preselected volume of a secondreactant, at a desired or preselected temperature for a desired orpreselected amount of time. The device then repeats the assay, butvaries at least one of the volume of the first or second reactants, thetemperature, or the amount of time allowed for the reaction. This isrepeated until a desired number of varied reactions are performed, i.e.,generating sufficient data to permit an estimation of optimal assayconditions which will produce an optimal result of the reaction, withina desired range of statistical significance. “optimal assay conditions”include those conditions that are required to achieve the desired resultof the reaction. Such desired results can include maximization ofreaction yields, but also includes assay conditions which are optimizedfor sensitivity to one variable, e.g., inhibitor concentration, and thelike.

An assay optimization using the microfluidic devices and systems of theinvention are illustrated through a competitive binding assay, e.g.,antibody-antigen binding. A microfluidic device for performing a bindingassay is illustrated in FIG. 4A. As shown, the microfluidic device 400is fabricated into a planar solid substrate 402. The device includes amain channel 404, which includes a separate reaction zone 404 a andseparation zone 404 b. The device also includes a sample well 406, afirst buffer well 408, an antigen well 410, an antibody well 412 and awaste well 414. Second buffer well 416 and waste well 428 are alsoincluded. The main channel 404 is linked to wells 406 through 412 viafluid channels 414-420, respectively. Wells 416 and 428 are linked tothe main separation channel 404 b via channels 422 and 424,respectively. Fluid direction within the device is carried outsubstantially as described herein, e.g., via the concommittentapplication of appropriate electrical voltages at multiple wells. Again,the device includes a detection zone 426 toward one end of the mainchannel, to allow detection of the labeled components as they move alongthe main channel.

In operation, the antibody panel to be screened against the sample isprovided as a mixture or cocktail, and placed in antibody well 412. Asimilar cocktail of the various different, labeled antigens for whichthe sample is being screened is placed in the antigen well 410. Labelingof antigens, or in some cases, antibodies, can be carried out by avariety of well known methods, and can include enzymatic, fluorescent,calorimetric, luminescent or other well known labeling techniques.

Initially, an antigen control is run. Specifically, antigen is pumpedfrom well 410 to waste well 428 via channels 418, 404 (through zones 404a) and 424. A measured fluid slug or region of labeled antigen is theninjected into and pumped along the main channel 404 and throughseparation zone 404 b. The labeled antigens electrophorese into theconstituent antigens, which are flowed past a detector 426. An exampleof data obtainable from the antigen control is shown in FIG. 4B, whereeach of the three peaks represents a different antigen in the antigencocktail. The peak heights for the antigen control are measured forlater use in quantification of the antigen in the sample. From therelative retention times, one can also determine that all of the labeledantigens are present in the cocktail.

Next, an antigen/antibody complex control is run. In particular,constant streams of antibody and antigen are pumped from theirrespective wells into the main channel 404, and particularly thereaction zone 404 a, and out to waste well 428. A measured slug of themixture is then injected into the separation channel 404 b. Complexationof the antigen with the antibody results in a shift in theelectrophoretic mobility of the labeled complex relative to that of thelabeled antigen alone. FIG. 4C represents data obtainable from theantigen/antibody complex control, where the three detected firstrepresent the uncomplexed labeled antigen, and the last three peaksrepresent the labeled antigen/antibody complex. Of course, in someaspects, electrophoretic mobility is affected in an opposite manner,i.e., resulting in a complex eluting faster than its constituentelements, and both contingencies are envisioned here. Concentrations ofantibody and labeled antigen will also generally be titrated to yieldresponses when contacted with the sample. Methods of titrating theseelements are well known in the art.

Finally, in a screening run, streams of antibody, antigen and sample areflowed continuously into the reaction channel 404 a and into waste well428. A slug of this mixture is then injected into the separation channel404 b. Any antigen of interest in the sample will compete for binding toits counterpart antibody with the corresponding labeled antigen,resulting in a reduction in the level of labeled complex, or an increasein the level of labeled, uncomplexed antigen. An example of dataobtainable from a test run is shown in FIG. 4D. As shown, the data wouldindicate that the sample contains an amount of antigen AG-1 and AG-3,but little or no AG-2. A quantitative determination of the levels ofthese various antigens within the sample can be obtained by comparingthe peak heights, either labeled, uncomplexed antigen, or labeledcomplex, from the test run to those of one or both of the control runs,where the difference (e.g., δ₁ and/or δ₂) is indicative of the amount ofantigen in the sample. See, e.g., Evangelista et al., Am. Clin. Lab.:27-28 (1995).

Additional wells and channels are optionally provided connected todifferent reagent injection channels, e.g., 414-422, to dilute thesevarious elements, in order to optimize the particular assay system.

Where different antigens, antibodies or complexes thereof, in a givenpanel screen lack sufficiently different electrophoretic mobilities, oneor more these elements are optionally chemically modified, e.g., by theaddition of charged groups, to alter the electrophoretic mobility ofthat element without substantially affecting that element's interactionwith other elements.

In performing the above-described assay format, a number of variableswould be expected to affect the assay performance. A number of thesevariables are set forth in Table 1, with a number of possible levels setforth for each variable.

TABLE 1 # of Variable Lev- Levels Sample Conc. els 3 low medium high %Ratio of 4 33:33:33 50:25:25 25:50:25 25:25:50 [Sample:Ag:Ab] Antibodytype 2 Vendor A Vendor B (vendor) Reaction Time 2 0.4 mm/s 0.8 mm/sReaction 2 25° C. 37° C. Temp. Injected 2 20 pl 50 pl Volume Separation2 0.4 mm/s 0.8 mm/s Time

As provided in this example, the assay has a total of 7 variables, eachof which has 2, 3 or 4 levels of variability. In order to perform a fullfactorial experiment covering these variables, 384 separate reactionruns would be required. Even assuming a ⅛ fractional factorialexperiment, 48 separate runs would be required, which when duplicated,would result in 96 separate runs. When performed at a bench scale, suchan experiment would take hours and would require substantial attentionfrom the investigator to ensure that each assay run is performedcorrectly and accurately. However, in the above described microfluidicformat, each run is automatically performed typically in approximately30 seconds per run. This would permit running all 48 distinct runs, induplicate, in less than one hour (in parallel microfluidic formats, asdiscussed below, the assay could easily be run in a few minutes).Further, the entire experiment is automatically controlled by thecomputer control system of the microfluidic system, as described herein.

After all of the assays are performed, the results are analyzed andoptimal assay conditions are determined. Analysis of these results istypically carried out in the control computer system using readilyavailable computer software, designed for experimental optimization,e.g., Design-Ease™ statistical optimization software.

Drug Screening Assays

In addition to sequencing, the integrated microfluidic system of theinvention is broadly useful in a variety of screening assays where theresults of mixing one or more components are to be determined, andparticularly, where the results determined are used to select additionalreagents to be screened.

As described more fully below, the integrated microfluidic system of theinvention can include a very wide variety of storage elements forstoring reagents to be assessed. These include well plates, matrices,membranes and the like. The reagents are stored in liquids (e.g., in awell on a microtiter plate), or in lyophilized form (e.g., dried on amembrane), and can be transported to an assay component of themicrofluidic device (i.e., a microfluidic substrate having reactionchannels or the like) using conventional robotics, or using anelectropipettor as described below.

Because of the breadth of the available sample storage formats for usewith the present invention, virtually any set of reagents can be sampledand assayed in an integrated system of the present invention. Forexample, enzymes and substrates, receptors and ligands, antibodies andligands, proteins and inhibitors, cells and growth factors orinhibitors, viruses and virus binding components (antibodies, proteins,chemicals, etc.) immunochemicals and immunoglobulins, nucleic acids andnucleic acid binding chemicals, proteins, or the like, reactantchemicals (acids, bases, organic molecules, hydrocarbons, silicates,etc.) can all be assayed using the integrated systems of the invention.For example, where a molecule which binds a protein is desired,potential binding moieties (chemicals, peptides, nucleic acids, lipids,etc.) are sequentially mixed with the protein in a reaction channel, andbinding is measured (e.g., by change in electrophoretic mobility,quenching of fluorescent protein residues, or the like). Thousands ofcompounds are easily screened using this method, in a short period oftime (e.g., less than an hour).

An advantage of the integrated nature of the present system is that itprovides for rational selection of structurally or functionallyhomologous compounds or components as the assay progresses. For example,where one compound is found to have binding activity, the selection of asecond compound to be tested can be performed based upon structuralsimilarity to the first active compound. Similarly, where a compound isshown to have activity in a cell (e.g., up-regulation of a gene ofinterest) at second compound affecting the same cellular pathway (e.g.,calcium or inositol phosphate second messenger systems, etc.) can beselected from the group of available compounds for testing. In this way,it is possible to focus screening assays from purely random at theoutset to increasingly focused on likely candidate compounds as theassays progress.

Further details on drug screening assays adaptable to the presentinvention are found in co-pending application U.S. Ser. No. 08/671,987.

Additional Nucleic Acid Analysis

Genomic material is subject to a certain amount of variation from oneindividual of a particular species to another. For example in amammalian genome of approximately 3 billion base pairs, approximately0.1%, or 3 million base pairs would be expected to vary amongindividuals, and a large number of these variations would be expected tobe linked to or result in potentially important traits.

A number of methods are currently available for identifying anddistinguishing these variations other than simply sequencing the nucleicacids as described above. For example, Kozal et al., Nature Medicine2(7):753-759 (1996), describes the use of high density oligonucleotideprobe arrays in identifying naturally occurring mutations in HIVinfected patients, which mutations augment resistance to particularantiviral agents, e.g., protease inhibitors.

Alternative methods for identifying these variations include actual DNAsequencing discussed above, oligonucleotide ligase assays, includingLCR, DNA polymerase based methods, and allele specific amplificationmethods. Although these methods are generally effective at benchtopscales when analyzing single or few loci, when comprehensive geneticinformation is desired, e.g., requiring analysis of large numbers ofloci, the conditions must be optimized for each locus, requiring theperformance of massive numbers of experiments, rendering such methodsoverly expensive, cumbersome and largely impractical.

In related aspects, the microfluidic devices and systems can be readilyused to perform nucleic acid analysis for identifying and mapping suchvariations, without the need for amplification or sequencing steps.Briefly, the particular assay system employs a hybridization of acomplex nucleic acid sample to groups of oligonucleotide probes that arecomplementary to different portions of the target sequence and that areimmobilized in different regions of a reaction channel. Enrichment of atarget nucleic acid of interest is carried out by the iterativehybridization, washing and release of the target from theseoligonucleotide probes. These probes are optionally complementary todifferent portions of the target or overlapping portions and they areoptionally the same or different lengths. A schematic illustration of adevice for such analysis is shown in FIG. 5A.

As shown, the device 500 is again fabricated into a solid substrate 502and includes a main analysis channel 504. The main analysis channelincludes first, second and third hybridization sites 506, 508 and 510,respectively. Each of reservoirs 512-520 are connected to the mainanalysis channel by a series of intersecting channels 522-536.

In operation, a sample containing a targeted nucleic acid is placed inreservoir 516. Where the sample includes double-stranded genomnic DNA,the sample is optionally denatured under basic conditions. This isaccomplished, e.g., by delivering a volume of dilute base, e.g., NaOH,from reservoir 514 via channel 524, to be mixed with the sample atintersection of channels 526 and 524. This intersection optionallycomprises a widened channel or chamber fabricated into the substrate, tofacilitate mixing of the sample and dilute base or to allow for morerefined control of reaction times. The denatured sample is then movedalong channel 528. The dilute base is optionally neutralized bydelivering an equal volume of similarly dilute acid, e.g., HCl, fromreservoir 512, via channel 522, to be mixed with the basic sample at theintersection of channels 522 and 528, which again, can comprise awidened channel or chamber design to facilitate mixing or to allow formore refined control of reaction times. Because samples will typicallyinclude highly complex nucleic acids, this complexity generally preventsthe sample from rapidly re-annealing. The neutralized, denatured sampleis then moved into the main analysis channel 504. Within the mainanalysis channel are hybridization sites 506, 508 and 510, at whichsites are immobilized short, synthetic oligonucleotides that arecomplementary to different portions of a target sequence. Immobilizationof oligonucleotides on solid substrates is optionally carried out by avariety of known methods. For example, often solid supports will includefunctional groups to which oligonucleotides are optionally coupled.Alternatively, substrates are optionally treated to provide such groups,e.g., by silanation of silica substrates.

These oligonucleotides comprise a set of sequences having homology tothe target sequence of interest, but not necessarily to each other,preferably of sequentially increasing lengths along the series of siteswithin the main channel 504, such as 10, 15, and 20 nucleotides inlength, at sites 506, 508 and 510, respectively. The lengths of theprobes generally varies depending upon the length and composition of thetarget sequence. The target sequence is preferably at least as long as,if not longer than the longest oligonucleotide. Typically, the probesare arranged in the reaction channel from lowest affinity to highestaffinity in the direction of flow for the gradient of denaturant. Targetsequence that dissociates from the first or weakest affinity probe willthen associate with the next probe in the series, and so on. Statedanother way, the lowest affinity probe will be located in the reactionchannel at a point nearest to the source of denaturant, and willtherefore receive the denaturant gradient first. Probes with strongeraffinity will be located sequentially further from the source ofdenaturant, with the probe having the strongest affinity being furthestfrom the source of denaturant.

Once directed into the reaction channel 504, the sample is presented tothe first group of probes 506, under conditions suitable forhybridization to those probes. By “conditions suitable forhybridization” is meant conditions of chemical composition, temperature,and the like, under which the target sequences are capable ofhybridizing to a particular probe sequence. “Stringent hybridization” inthe context of these nucleic acid hybridization experiments are sequencedependent, and are different under different environmental parameters.Generally, highly stringent hybridization conditions are selected to beabout 5°-15° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and ph. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget RNA sequence hybridizes to a perfectly matched oligonucleotideprobe. Very stringent conditions are selected to be nearly equal to theT_(m) for a particular probe (e.g., 0°-5° C. below the meltingtemperature). An oligonucleotide “specifically hybridizes” to aparticular target when the probe hybridizes with a least twice thesignal intensity of a control probe. Where the control probe differs byless than 10% (often by only 1 nucleotide) from a test probe, the testprobe is an “allele-specific” probe (to indicate that the test probe canbe used to distinguish between two different alleles of a target whichdiffer by a single nucleotide). See also, Gait, ed. OligonucleotideSynthesis: A Practical Approach, IRL Press, Oxford (1984); W. H. A.Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K. L. Dueholm J.Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methods in MolecularBiology, volume 20; and Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology—hybridization with nucleic acidprobes, e.g., part I chapter 2 “overview of principles of hybridizationand the strategy of nucleic acid probe assays”, Elsevier, N.Y. for abasic guide to nucleic acid hybridization.

Sequential purification of the target portion of the genome can beachieved by sequential selective hybridizations to theseoligonucleotides. Thus, for example, when the sequence of interest is 20nucleotides or longer in length, one would expect that a shorteroligonucleotide, such as a 10-mer, will hybridize to many more sites inthe genome than merely the target sequence, just on the statisticalbasis of any particular sequence of 10 nucleotides appearing in thegenome. These sequences will hybridize to the 10-mer oligonucleotide,while non-hybridizing DNA can be washed out of the pool via wastereservoir 538 with diluent in reservoir 518 supplied through channels532 and 536. The reduced pool, which is actually enriched for sequenceshybridizing to the 10-mer, is then subjected to a gradient of denaturantwhich is delivered from reservoir 520. Useful denaturants include thosealready described herein, including, e.g., formamide, and the like. Themaximal concentration of the denaturant is calibrated to maintain amaximal stability of the target sequence/10-mer duplex, therebyeliminating imperfectly hybridized target sequences from the 10-mer orother probes, including double base and single base mismatchedprobe/target hybrids. Determination of optimal levels of denaturant isgenerally carried out experimentally, i.e., by determining optimalhybridizan conditions for a given probe sequence.

Denaturant is transported from reservoir 520 through channels 534 and536, while diluent can be added from reservoir 518 through channel 532to the intersection of channels 532 and 534. Complexity of the samplenucleic acids is substantially reduced by this step. For example, atypical mammalian genome having over 10⁵ base pairs, would be expectedto have approximately 10³ sites capable of hybridizing to a 10-merprobe, effectively allowing a one million-fold reduction in samplecomplexity.

Following removal of less strongly bound species, the denaturantgradient is restored to a level that causes dissociation of the targetfrom the 10-mer probes, but which permits hybridization to the 15-meroligonucleotide. As a particular sequence of 15 nucleotidesstatistically occurs with less frequency than a 10 nucleotide sequence,again the complexity of the sample DNA will be reduced whennon-hybridized DNA is washed out of the main analysis channel 504. Theseenrichment steps can be performed with oligonucleotides of increasinglength until the desired level of enrichment is achieved. In someembodiments, oligonucleotide probes need not be of increasing length;multiple steps using different oligonucleotide probes will continue toenrich the DNA population for the sequence of interest based onprobability of the oligonucleotide sequence occurring in populations ofdecreasing complexity.

Preferably, at least one enrichment step is performed beforehybridization to oligonucleotides that “type” the target sequence forthe presence of a particular target sequence or variation.

Although described in terms of use of chemical denaturants, it will bereadily appreciated that other chemical or non-chemical treatments areoptionally used to vary the hybridization conditions, includingadjusting pH, temperature. Similarly, although varied affinity among theprobes is generally described as being carried out by use of differentlength probes, it will also be understood that different probecompositions can also be used to vary affinity of the probe to thetarget. For example, G-C rich probes will hybridize with greateraffinity, i.e., have a higher melting temperature, than A-T rich probes.These chemical properties can be exploited in practicing this aspect ofthe invention.

Finally, target nucleic acid typed by virtue of its enrichment andsubsequent hybridization to a higher affinity probe, e.g., a 15-mer or20-mer, can be released from the final hybridization site and flowedalong the analysis channel 504. The “typed” target sequence is flowedpast a detection window 540, whereupon it can be detected, i.e., byvirtue of an incorporated labelling group. A variety of direct andindirect labeling and detection methods are well known for nucleicacids, including radiolabeling methods, fluorescent labeling, eitherdirectly or from an intercalating fluorescent dye, chemiluminescentlabeling, colorimetric labeling, labeling with ligands or anti-ligands,e.g., biotin/avidin or streptavidin, and the like.

In an alternate method, the target sequence can be identified bydetecting the accumulation of the detectable label at the finalhybridization or “typing” site, following the final washing step.

Melting Point Analysis of Nucleic Acids

In a similar embodiment, the systems, devices and methods of the presentinvention, can be used to detect variations in nucleic acid sequences bydetermining the strength of the hybridization between the targetednucleic acid and probes that are putative perfect complements to thetarget. By identifying the difference in stability between the imperfectand perfect hybrids under conditions of increasing hydrogen bond stress,one can identify those nucleic acids that contain a variation.

In practice, a microfluidic device is configured to accept a samplecontaining an amplified nucleic acid or polynucleotide sequence ofinterest, convert it to single-stranded form, facilitate hybridizationwith a nucleic acid probe, such as an oligonucleotide, and then subjectthe hybridization mixture to a chemical or temperature gradient thatdistinguishes between perfectly matched targets and those that differ byat least one base pair (mismatch). In some embodiments, one or more locior targeted areas of the sample polynucleotide are first amplified bysuch techniques as PCR or sandwich hybridization. In other embodiments,unamplified polynucleotide is provided to the device and amplifiedtherein, such as in the non-thermal amplification embodiments describedbelow.

A schematic illustration of a microfluidic device for carrying out thisanalysis is shown in FIG. 5B employing the same schematic layout as thedevice shown in FIG. 5A. In this aspect, a sample containing a nucleicacid is introduced into sample well 516. This sample is, e.g.,introduced into the device, preamplified, or it can be transported towell 516 from another portion of the device where the nucleic acid wasamplified, e.g., in integrated operations. Thus, although shown as awell, sample well 516 can be a reservoir or an inlet supplied by anexternal reservoir or separate reaction chamber. In some embodiments,when the polynucleotide is amplified, one end of the amplifiedoligonucleotide (e.g., PCR product) is terminated with groups such asphosphorothioate bonds that prevent exonucleolytic action by enzymessuch as T7 DNA polymerase.

A preselected amount of amplified target is then fluidically movedthrough channel 526. A preselected amount of exonuclease, e.g., T7 DNApolymerase, placed in well 514, is concurrently moved along channel 524.Where channel 526 and 524 intersect (intersection 525), the target andthe exonuclease mix in channel 528, and the target is subjected toenzymatic digestion to render the target single-stranded. Alternatively,single stranded target is optionally prepared by asymmetric PCR.

The resulting single-stranded molecules are then moved along channel 528to the intersection of this channel and probe channel 522. Probe well512 contains oligonucleotide probes which are putatively complementaryto a region of the target which contains a potential variation. Theprobe containing solution is delivered along probe channel 522 to theintersection 523 with channel 528, whereupon the probe solution mixesand hybridizes with the single stranded target. As above, a widenedchannel or chamber is optionally provided at these intersections tofacilitate mixing of the materials.

Hybridization of the probe results in a perfect hybrid with nomismatches when the sample polynucleotide contains the complementarysequence, i.e., no variation, or in a hybrid with mismatches if thesample polynucleotide differs from the probe, i.e., contains a sequencevariation. The stability of the imperfect hybrid differs from theperfect hybrid under conditions of increasing hydrogen bond stress. Avariety of methods are available for subjecting the hybrids toincreasing hydrogen bond stress, sufficient to distinguish betweenperfectly matched probe/target hybrids and imperfect matches. Forexample, the hybrids are optionally subjected to a temperature gradient,or alternatively, can be subjected to increasing concentrations of achemical denaturant, e.g., formamide, urea, and the like, or increasingpH.

As shown, the hybridized target/probe mixture is moved through channel530 to the intersection 531 of this channel with denaturant channel 536.Denaturant, placed in denaturant well 520 is concurrently delivered tointersection 531, whereupon it mixes with the target/probe hybrid. Thedenaturant can be diluted with an appropriate diluent buffer suppliedfrom diluent well 518 via channel 532. The differences in stability ofthe hybrids under denaturing conditions can be detected by an integratedseparation column such as a capillary channel 528 where molecularsieving can be done.

Following mixing with the denaturant, hybridized and nonhybridizednucleic acids are electrophoretically separated by moving the mixturealong separation channel 504. The separation channel can include any ofa number of separation matrices, e.g., agarose, polyacrylamide,cellulose, or the like.

The assay is then repeated several times, varying the concentration ofdenaturant with each successive assay. By monitoring the level of hybridor single stranded target, one can determine the concentration ofdenaturant at which the probe-target hybrid is denatured. This level isthen compared to a standard curve, to determine whether one or morevariations are present.

Microfluidic Detection Apparatus

The microfluidic apparatus of the invention often, though notnecessarily, comprise a substrate in which fluidic reagents, mixtures ofreagents, reactants, products or the like are mixed and analyzed. A widevariety of suitable substrates for use in the devices of the inventionare described in U.S. Ser. No. 08/761,575, U.S. Pat. No. 6,046,056entitled “High Throughput Screening Assay Systems in Microscale FluidicDevices” by Parce et al. A microfluidic substrate holder is optionallyincorporated into the devices of the invention for holding and/or movingthe substrate during an assay. The substrate holder optionally includesa substrate viewing region for analysis of reactions carried out on thesubstrate. An analyte detector mounted proximal to the substrate viewingregion to detect formation of products and/or passage of reactants alonga portion of the substrate is provided. A computer, operably linked tothe analyte detector, monitors formation of reactants, separation ofsequencing products, or the like. An electrokinetic component typicallyprovides for movement of the fluids on the substrate. Microfluidicdevices are also described in U.S. Ser. No. 08/691,632.

A principal component of nucleic acid analysis is molecular partition.Channels in microfluidic substrates can be used for molecularseparations. In addition, the dexterous fluidics in the microfluidicdevices herein produce exquisite control over injection volume—aprincipal parameter determining resolution in molecular partitioning.Aside from biochemistry and analytical capabilities in microdevices,systems that automate access to reagents and specimens are highly usefulfor the integrated systems herein. In high throughput pharmaceuticalscreening a “world-to-chip” interface capable of importing samples fromconventional liquid vessels (such as test tubes or 384-well plates), orfrom solid dots of reagent on substrates is useful. The ability toimport 1000's of different samples with inter-sample intervals as shortas 5 seconds is achieved using the systems herein. A simple system willperform experiments at the rate of 10,000 experiments per channel perday. Simple parallelization of the channels produces a capacity of morethan 1 million such assays per instrument-day.

Accordingly, in one embodiment, a “sipping” strategy for introducingsolubilized reagents or samples into a microfluidic substrate from astandard microplate is used. This is adapted to elements of nucleicacids testing, for example to allow for random access to a library ofprobes or primers. Although this technology works, the advantage ofreagent economy that is a hallmark of the microfluidic technology issomewhat nullified when a chemical library must be presented to thesystem in tens of microliter volumes, e.g., in microplates.

In order to take advantage of the very small quantities of reagentsrequired by the chip, and to make a system scalable to millions ofexperiments, a solid phase reagent interface uniquely suited to highthroughput LabChip processing is desirable. Several new interfaces thatmake use of reagents dried in microarrays on a solid surface aredescribed herein. These configurations are suited to the needs ofdiagnostic products in which elements need to be standardized,convenient, and have acceptable shelf-life. Many robotic systems are nowavailable that can deposit arrays of individual solutions at highdensities (1000 per square centimeter and greater). These are typicallyused as capture elements in heterogeneous phase biochemical assays suchas nucleic acids hybridization. The same approach can be used to depositelements of solution phase reactions (PCR primers, probes, sequencingprimers, etc.). Using these approaches, systems that access solid phasereagents at densities of up to 1000 spots per square centimeter aremade.

As described above, a preferred integrated method of the inventionincorporates the use of pre-synthesized sets of primers for sequencingand/or PCR, and or reagents to be tested in drug screening assays. Adevice of the invention preferably includes a primer storage and/orprimer transport mechanism for delivering selected primers to a reactionchannel in the microfluidic device. Exemplary storage mechanismsoptionally include components adapted to holding primers in a liquid orlyophilized form, including containers, containers with separatecompartments, plates with wells (e.g., small microtiter plates withhundreds or thousands of wells) membranes, matrices, arrays of polymers,or the like. Additional embodiments for handling dried reagents on solidsubstrates are shown below.

As discussed above, the region for storage of the primers is optionallylocated on the substrate of the microfluidic device in fluid connectionto a mixing region or channel on the substrate in which a biochemicalreaction (PCR, sequencing or the like) is carried out. In an alternativeembodiment, the primer storage area is physically separated from thesubstrate. In this embodiment, the primers can be loaded onto thesubstrate, either manually, or using an automated system. For example, aZymate XP (Zymark Corporation; Hopkinton, Mass.) automated robot using aMicrolab 2200 (Hamilton; Reno, Nev.) pipetting station can be used totransfer parallel samples to regularly spaced wells in a manner similarto transfer of samples to microtiter plates. If the primers are storedin lyophilized form (e.g., dried on a substrate), a portion of thelyophilized primer is typically suspended in an aqueous solution tofacilitate transfer to a microfluidic substrate. An electropipettor asdescribed above can be used to select and transport samples to amicrofluidic substrate from a well plate, or from any region of amicrofluidic substrate. Because integration of the electropipettor withthe microfluidic substrates of the invention is relatively simple,electropipettor embodiments are preferred.

In preferred embodiments including an electropipettor, a variety ofstorage systems for storing reagents, such as primers for delivery tothe microfluidic devices of the invention, are applicable. Compounds areconveniently sampled with the electropipettor from well plates, or fromimmobilized samples stored on a matrix (e.g., a porous, hydrophilic, orhydrophobic matrix), or from dried samples stored on a substrate such asa nitrocellulose, nylon or nytran membrane. In embodiments where thesamples are dried, the samples are solubilized using theelectropipettor, which can be operated to expel a small volume of fluidonto the dried reagent, followed by pipetting the expelled fluidcomprising the reagent into the electropipettor. See also, U.S. Ser. No.08/671,986.

Accordingly, the present invention provides sampling systems whichprovide the compounds to be sampled in an immobilized format on amembrane matrix or the like, i.e., that the sample material is providedin a fixed position, either by incorporation within a fixed matrix,e.g., a porous matrix, a charged matrix, a hydrophobic or hydrophilicmatrix, or the like, which maintains the sample in a given location.Alternatively, such immobilized samples include samples spotted anddried upon a given sample matrix. In preferred aspects, the compounds tobe screened are provided on a sample matrix in dried form. Typically,such sample matrices will include any of a number of materials that canbe used in the spotting or immobilization of materials, including, e.g.,membranes, such as cellulose, nitrocellulose, PVDF, nylon, polysulfoneand the like. Typically, flexible sample matrices are preferred, topermit folding or rolling of the sample matrices which have largenumbers of different sample compounds immobilized thereon, for easystorage and handling.

Generally, samples are optionally applied to the sample matrix by any ofa number of well known methods. For example, sample libraries arespotted on sheets of a sample matrix using robotic pipetting systemswhich allow for spotting of large numbers of compounds. Alternatively,the sample matrix is treated to provide predefined areas for samplelocalization, e.g., indented wells, or hydrophilic regions surrounded byhydrophobic barriers, or hydrophobic regions surrounded by hydrophilicbarriers (e.g., where samples are originally in a hydrophobic solution),where spotted materials will be retained during the drying process. Suchtreatments then allow the use of more advanced sample applicationmethods, such as those described in U.S. Pat. No. 5,474,796, wherein apiezoelectric pump and nozzle system is used to direct liquid samples toa surface. Generally, however, the methods described in the '796 patentare concerned with the application of liquid samples on a surface forsubsequent reaction with additional liquid samples. However, thesemethods are readily modified to provide dry spotted samples on asubstrate. Similarly, the use of ink-jet printing technology to printbiological and chemical reagents onto substrates is well developed. See,e.g., Wallace (1996) Laboratory Automation News 1(5):6-9 where ink-jetbased fluid microdispensing for biochemical applications is described.

Similarly, cleavable linkers attaching compounds to an array can be usedto store the compounds in an array, followed by cleavage from the array.A variety of cleavable linkers, including acid cleavable linkers, lightor “photo” cleavable linkers and the like are known in the art. Exemplararrays are described in Pirrung et al., U.S. Pat. No. 5,143,854 (seealso, PCT Application No. WO 90/15070), Fodor et al., PCT PublicationNo. WO 92/10092 Fodor et al. (1991) Science, 251: 767-777; Sheldon etal. (1993) Clinical Chemistry 39(4): 718-719; Kozal et al. (1996) NatureMedicine 2(7): 753-759 and Hubbell U.S. Pat. No. 5,571,639.Immobilization of assay components in an array is typically be via acleavable linker group, e.g., a photolabile, acid or base labile linkergroup. Accordingly, the assay component is typically released from theassay e.g., by exposure to a releasing agent such as light, acid, baseor the like prior to flowing the test compound down the reactionchannel. Typically, linking groups are used to attach polymers or otherassay components during the synthesis of the arrays. Thus, preferredlinkers operate well under organic and/or aqueous conditions, but cleavereadily under specific cleavage conditions. The linker is optionallyprovided with a spacer having active cleavable sites. In the particularcase of oligonucleotides, for example, the spacer is selected from avariety of molecules which can be used in organic environmentsassociated with synthesis as well as aqueous environments, e.g.,associated with nucleic acid binding studies. Examples of suitablespacers are polyethyleneglycols, dicarboxylic acids, polyamines andalkylenes, substituted with, for example, methoxy and ethoxy groups.Linking groups which facilitate polymer synthesis on solid supports andwhich provide other advantageous properties for biological assays areknown. In some embodiments, the linker provides for a cleavable functionby way of, for example, exposure to an acid or base. Additionally, thelinkers optionally have an active site on one end opposite theattachment of the linker to a solid substrate in the array. The activesites are optionally protected during polymer synthesis using protectinggroups. Among a wide variety of protecting groups which are useful arenitroveratryl (NVOC) α-methylnitroveratryl (Menvoc), allyloxycarbonyl(ALLOC), fluorenylnethoxycarbonyl (FMOC),α-methylnitro-piperonyloxycarbonyl (MeNPOC), -NH-FMOC groups, t-butylesters, t-butyl ethers, and the like. Various exemplary protectinggroups are described in, for example, Atherton et al., (1989) SolidPhase Peptide Synthesis, IRL Press, and. Greene, et al. (1991)Protective Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, NewYork, N.Y.

Other immobilization or spotting methods are similarly employed. Forexample, where samples are stable in liquid form, sample matrices caninclude a porous layer, gel or other polymer material which retain aliquid sample without allowing excess diffusion, evaporation or thelike, but permit withdrawal of at least a portion of the samplematerial, as desired. In order to draw a sample into an electropipettor,the pipettor will free a portion of the sample from the matrix, e.g., bydissolving the matrix, ion exchange, dilution of the sample, and thelike.

Whether the storage substrate is a filter, membrane, microtiter plate orother material holding reagents of interest, the substrate canconveniently be moved using, a mechanical armature. Typically, thespatial location (or “physical address”) of the reagents on thesubstrate are known. The armature moves the substrate relative to themicrofluidic substrate (and electropipettor, where applicable) so thatthe component for transferring reagent from the substrate to thechannels and wells of a microfluidic substrate (e.g., anelectropipettor) contacts the desired reagent. Alternatively, themicrofluidic substrate or electropipettor can be moved by an armaturerelative to the storage substrate to achieve the same effect. Similarly,both the storage substrate and the microfluidic substrate can be movedby the mechanical armature to achieve the same effect. In anotheraspect, the microfluidic substrate, storage substrate or transferringcomponent (e.g., electropipettor) can be manually manipulated by theoperator.

A variety of electropipettors, including “resolubilization” pipettorsfor solubilizing dried reagents for introduction into microfluidicapparatus are described in Ser. No. 08/671,986, supra. In brief, anelectropipettor pipettor having separate channels is fluidly connectedto an assay portion of the microfluidic device (i.e., a microfluidicsubstrate having the reaction and/or analysis and/or separationchannels, wells or the like). In one typical embodiment, theelectropipettor has a tip fluidly connected to a channel underelectroosmotic control. The tip optionally includes features to assistin sample transfer, such as a recessed region to aid in dissolvingsamples. Fluid can be forced into or out of the channel, and thus thetip, depending on the application of current to the channel. Generally,electropipettors utilize electrokinetic or “electroosmotic” materialtransport as described herein, to alternately sample a number of testcompounds, or “subject materials,” and spacer compounds. The pipettorthen typically delivers individual, physically isolated sample or testcompound volumes in subject material regions, in series, into the samplechannel for subsequent manipulation within the device. Individualsamples are typically separated by a spacer region of low ionic strengthspacer fluid. These low ionic strength spacer regions have highervoltage drop over their length than do the higher ionic strength subjectmaterial or test compound regions, thereby driving the electrokineticpumping, and preventing electrophoretic bias. On either side of the testcompound or subject material region, which is typically in higher ionicstrength solution, are fluid regions referred to as first spacer regions(also referred to as high salt regions or “guard bands”), that contactthe interface of the subject material regions. These first spacerregions typically comprise a high ionic strength solution to preventmigration of the sample elements into the lower ionic strength fluidregions, or second spacer region, which would result in electrophoreticbias. The use of such first and second spacer regions is described ingreater detail in U.S. patent application Ser. No. 08/671,986, supra.Spacers are not, however, required, particularly in those embodimentswhere transported components such as primers have the same charge andmass. It will be appreciated that embodiments using identically (ornearly identically) sized primers, such as modular primers, can be usedwithout guard bands.

In FIG. 6A and FIG. 6B, two solid phase samplers are shown, depictingtwo approaches for accessing dried reagent arrays by microfluidicapparatus. FIG. 6A shows micromachined chip 605 having three capillarychannels 610, 615, and 620. The channels terminate at one end of chip605 in sample cup 625. Due to the differences in ionic strength of thesolution in channels 610, 615, and 620, application of a potential fromchannel 610 to the channel 620, will force fluid into sample cup 625where it can dissolve dried reagent 630. Subsequent application of apotential from right channel 610 to central channel 620 will drawsolubilized reagent 635 into central channel 620. In FIG. 6B, poroussubstrate (e.g. microchannel alumina) 640 contains dried reagents 645.Application of a sufficient voltage from bottom solvent-supply capillary650 to chip capillary 655 attached to a microfluidic element (e.g., achannel on a chip; not shown) causes fluid to pass through poroussubstrate 640 and into capillary 655 attached to the microfluidicelement. In passing through substrate 640, the fluid dissolves driedreagent 645 and then carries it into the microfluidic element. In bothsystems, substrate 640 is moved, e.g., by robot to position the samplingcapillary over the appropriate reagent site.

Alternatively, in embodiments omitting an electropipettor, the channelsare individually fluidly connected to a plurality of separate reservoirsvia separate channels. The separate reservoirs each contain a separatetest analyte with additional reservoirs being provided for appropriatespacer compounds. The test compounds and/or spacer compounds aretransported from the various reservoirs into the sample channels usingappropriate fluid direction schemes. In either case, it generally isdesirable to separate the discrete sample volumes, or test compounds,with appropriate spacer regions.

One of skill will immediately recognize that any, or all of thecomponents of a microfluidic device of the invention are optionallymanufactured in separable modular units, and assembled to form anapparatus of the invention. See also, U.S. Ser. No. 08/691,632, supra.In particular, a wide variety of substrates having different channels,wells and the like are typically manufactured to fit interchangeablyinto the substrate holder, so that a single apparatus can accommodate,or include, many different substrates adapted to control a particularreaction. Similarly, computers, analyte detectors and substrate holdersare optionally manufactured in a single unit, or in separate moduleswhich are assembled to form an apparatus for manipulating and monitoringa substrate. In particular, a computer does not have to be physicallyassociated with the rest of the apparatus to be “operably linked” to theapparatus. A computer is operably linked when data is delivered fromother components of the apparatus to the computer. One of skill willrecognize that operable linkage can easily be achieved using eitherelectrically conductive cable coupled directly to the computer (e.g.,parallel, serial or modem cables), or using data recorders which storedata to computer readable media (typically magnetic or optical storagemedia such as computer disks and diskettes, CDs, magnetic tapes, butalso optionally including physical media such as punch cards, vinylmedia or the like).

Microfluidic Substrates and Electrokinetic Modulators

Suitable microfluidic substrate materials are generally selected basedupon their compatibility with the conditions present in the particularoperation to be performed by the device. Such conditions can includeextremes of pH, temperature, salt concentration, and application ofelectrical fields. Additionally, substrate materials are also selectedfor their inertness to critical components of an analysis or synthesisto be carried out by the device.

Examples of useful substrate materials include, e.g., glass, quartz andsilicon as well as polymeric substrates, e.g. plastics. In the case ofconductive or semi-conductive substrates, it is occasionally desirableto include an insulating layer on the substrate. This is particularlyimportant where the device incorporates electrical elements, e.g.,electrical fluid direction systems, sensors and the like. In the case ofpolymeric substrates, the substrate materials are optionally rigid,semi-rigid, or non-rigid, opaque, semi-opaque or transparent, dependingupon the use for which they are intended. For example, devices whichinclude an optical, spectrographic, photographic or visual detectionelement, will generally be fabricated, at least in part, fromtransparent materials to allow, or at least, facilitate that detection.Alternatively, transparent windows of, e.g., glass or quartz, areoptionally incorporated into the device for these types of detectionelements. Additionally, the polymeric materials optionally have linearor branched backbones, and can be crosslinked or non-crosslinked.Examples of particularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate, PMMAs and the like.

In certain embodiments, the microfluidic substrate includes one or moremicrochannels for flowing reactants and products. At least one of thesechannels typically has a very small cross sectional dimension, e.g., inthe range of from about 0.1 μm to about 500 μm. Preferably thecross-sectional dimensions of the channels is in the range of from about1 to about 200 μm and more preferably in the range of from about 0.1 toabout 100 μm, often in the range of about 1 to 100 μm. In particularlypreferred aspects, each of the channels has at least one cross-sectionaldimension in the range of from about 0.1 μm to about 100 μm. It will beappreciated that in order to maximize the use of space on a substrate,serpentine, saw tooth or other channel geometries, are optionally usedto incorporate longer channels on less substrate area, e.g., tofacilitate separation of reaction products or reactants. Substrates areof essentially any size, with area typical dimensions of about 1 cm² to10 cm².

In general, the microfluidic devices will include one or more chambers,channels or the like, fluidly connected to allow transport of fluidamong the chambers and/or channels of these devices. By “microfluidic”is generally meant fluid systems, e.g., channels, chambers and the like,typically fabricated into a solid typically planar substrate, andwherein these fluid elements have at least one cross-sectional dimensionin the range of from about 0.1 to about 500 μm. Typically, the crosssectional dimensions of the fluid elements will range from about 1 μm toabout 200 μm. The term “channel” is defined above. A “chamber” willtypically, though not necessarily, have a greater volume than a channel,typically resulting from an increased cross-section having at least onedimension from about 10 to about 500 μm, although, as for channels, therange can span, e.g., 0.1 to about 500 μm. Although generally describedin terms of channels and chambers, it will generally be understood thatthese structural elements are interchangeable, and the terms are usedprimarily for ease of discussion. By “fluidly connected” is meant ajunction between two regions, e.g., chambers, channels, wells etc.,through which fluid freely passes. Such junctions may include ports orchannels, which can be clear, i.e., unobstructed, or can optionallyinclude valves, filters, and the like, provided that fluid freely passesthrough the junction when desired.

Manufacturing of these microscale elements into the surface of thesubstrates is generally carried out by any number of microfabricationtechniques that are known in the art. For example, lithographictechniques are employed in fabricating, e.g., glass, quartz or siliconsubstrates, using methods well known in the semiconductor manufacturingindustries such as photolithographic etching, plasma etching or wetchemical etching. See, Sorab K. Ghandi, VLSI Principles: Silicon andGallium Arsenide, NY, Wiley (see, esp. Chapter 10). Alternatively,micromachining methods such as laser drilling, air abrasion,micromilling and the like are employed. Similarly, for polymericsubstrates, well known manufacturing techniques are used. Thesetechniques include injection molding or stamp molding methods wherelarge numbers of substrates are produced using, e.g., rolling stamps toproduce large sheets of microscale substrates or polymer microcastingtechniques where the substrate is polymerized within a micromachinedmold. Polymeric substrates are further described in Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 Ser. No.08/843,212 filed Apr. 14, 1997 now U.S. Pat No. 5,885,470.

In addition to micromachining methods, printing methods are also used tofabricate chambers channels and other microfluidic elements on a solidsubstrate. Such methods are taught in detail in U.S. Ser. No. 08/987,803by Colin Kennedy, filed Dec. 10, 1997 entitled “Fabrication ofMicrofluidic Circuits by Printing Techniques.” In brief, printingmethods such as ink-jet printing, laser printing or other printingmethods are used to print the outlines of a microfluidic element on asubstrate, and a cover layer is fixed over the printed outline toprovide a closed microfluidic element.

The substrates will typically include an additional planar element whichoverlays the channeled portion of the substrate, enclosing and fluidlysealing the various channels. Attaching the planar cover element isachieved by a variety of means, including, e.g., thermal bonding,adhesives or, in the case of certain substrates, e.g., glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. The planar cover element can additionally beprovided with access ports and/or reservoirs for introducing the variousfluid elements needed for a particular screen, and for introducingelectrodes for electrokinetic movement.

Typically, an individual microfluidic device will have an overall sizethat is fairly small. Generally, the devices will have a square orrectangular shape, but the specific shape of the device can be easilyvaried to accommodate the users needs. While the size of the device isgenerally dictated by the number of operations performed within a singledevice, such devices will typically be from about 1 to about 20 cmacross, and from about 0.01 to about 1.0 cm thick.

Serial to Parallel Conversion

In performing a large number of parallel fluid manipulations, it isoften necessary to allocate a single fluid volume among several separatechannels or chambers for reaction or analysis. For example, a singlesample volume is introduced into a device along a single channel. Toperform a panel of desired screens on the sample, or perform the samescreen multiple times, it is necessary to direct portions of the sampleto separate reaction chambers or channels. Similarly, a series ofdiscrete and different sample volumes is individually directed from thesample introduction channel into multiple separate channels. Thisallocation or direction of a single fluid volume or multiple discretefluid volumes from a serial orientation, i.e., a single channel orchamber, to a parallel orientation, i.e., to multiple separate channelsor chambers, is termed “serial to parallel conversion.” This conversionis particularly applicable to the present invention, in which multipleassays can be run in parallel in a first assay screen, the resultsdetected (in serial or parallel) and a second series of parallel assaysselected for a second screen based upon the results of the first screen.

As applied to the present invention, methods of performing fluidicoperations that include a plurality of parallel fluid manipulations toprovide parallel fluidic analysis of sample materials is thereforeprovided, as are related apparatus. In the methods a microfluidic deviceis provided. The device has at least a first transverse reagentintroduction channel fluidly connected to a source of at least onereagent and a source of at least one sample material. The transversechannel is fluidly connected to a plurality of parallel reagent reactionchannels. A first reagent or mixture of reagents is selected from thesource of at least one reagent, and the first reagent is transportedthrough the reagent introduction channel and a portion of the reagent isaliqouted as described into at least one parallel reagent reactionchannel (typically into several parallel reaction chambers or channels).A first sample material is selected from the source of at least onesample material and the first sample material is aliquoted into at leasta first of the plurality of parallel reagent reaction channels. At leastone additional sample material, or at least one additional reagent isselected, and the additional sample material or additional reagent isaliquoted into at least a second of the plurality of parallel reagentreaction channels. The first sample material and the first reagent arecontacted in the first reagent reaction channels, causing a reaction ofthe first sample material and the first reagent. The at least oneadditional sample material or at least one additional reagent iscontacted with one or more fluid component such as the first samplematerial, the first reagent, at least one additional reagent, at leastone additional sample material, a second additional reagent, a secondadditional sample material or the like. The first reaction product ofthe first sample material and the first reagent is detected, as is asecond reaction product of at least one additional sample material or atleast one additional reagent and one or more fluid component (i.e., fromtwo parallel reactions in two or more parallel reaction channels). Basedupon the first or second reaction product, a secondary reagent and asecondary sample material are selected and the process repeated on thesesecondary components. It will be appreciated that this “parallelization”of multiple assays and selection of additional assays based upon theresults of a first series of assays can dramatically speed selection andperformance of related assays, e.g., in a drug screening, assayoptimization, diagnostic or nucleic acid sequencing context.

In one aspect, the method comprises parallel analysis of a plurality ofsample materials in the parallel channels, in which multiple reagentsare mixed in a plurality of the parallel channels with multiple samplematerials to form a multiple of products, and, based upon detection ofthe multiple products, selecting the secondary sample material andsecondary reagent. This multiply parallel format can additionally speedassay development and data acquisition. In one aspect, the microfluidicdevice includes the first transverse reagent introduction channel and atleast a second transverse channel, and a plurality of parallel channelsintersecting both of the first and second transverse channels. In thisformat, the step of aliquoting the portion of the reagent into at leastone parallel reagent reaction channel is performed by applying a firstvoltage across the first transverse reagent introduction channel and thesecond transverse channel to draw the portion of the reagent into thefirst transverse reagent introduction channel, whereby the portion ofthe reagent is present at intersections of the first channel and each ofthe plurality of parallel channels; and, applying a second voltage fromthe first transverse channel to the second transverse channel, whereby acurrent in each of the parallel channels is equivalent, and whereby theportion of the reagent at the intersections of the first transversechannel and each of the plurality of parallel channels is moved in toeach of the plurality of parallel channels.

In a second serial to parallel conversion aspect, methods of performinga plurality of separate assays on a single sample are provided. In thesemethods a microfluidic device having at least a first transverse channelfluidly connected to at least a source of the sample, a plurality ofseparate parallel channels fluidly connected to the first transversechannel, each of the separate channels having disposed therein reagentsfor performing a different diagnostic assay, and a fluid directionsystem for concurrently directing a portion of the sample into each ofthe plurality of parallel channels is provided. A portion of the sampleis transported into each of the parallel channels, whereby the sampleand the reagents disposed in the channel undergo a reaction. A result ofthe reaction of the sample and the reagents disposed in the channel, foreach of the parallel channels is detected.

Thus, in certain aspects, the devices and systems of the presentinvention generally include novel substrate channel designs to ensureflow of appropriate amounts of fluids in parallel channels, and therebyfacilitate serial to parallel conversion of fluids in these microfluidicdevices.

Serial to parallel conversion of fluids within a microfluidic device isimportant for a number of reasons. For example, where one is performinga number of separate analyses on a single sample, serial to parallelconversion can be used to aliquot the single sample among a number ofseparate assay channels in a microfluidic device. Alternatively, anumber of physically discrete and different samples, e.g., drugcandidates, diagnostic samples, or the like, are serially introducedinto a single device and allocated among a number of different parallelchannels subjecting the samples to the same or different analyses.

Schematic illustrations of serial to parallel conversions are shown inFIGS. 7A-7D. For example, in FIG. 7A, a single sample fluid region (701)is shown being converted to a plurality of separate aliquots of thesample fluid, in a series of parallel channels. Alternatively, as shownin FIG. 7B, separate aliquots of the same sample fluid, provided in aserial orientation in a single channel are allocated to each of severalseparate parallel channels. In a particularly useful aspect, as shown inFIG. 7C, a plurality of different compounds (701, 702, 703 and 704) areserially introduced into a microfluidic channel (top) and then are eachredirected to a separate parallel channel for separate analysis orfurther manipulation. FIG. 7D also illustrates a particularly usefulapplication of serial to parallel conversion where a plurality ofdifferent samples (701, 702, 703 and 704) are serially introduced into amicrofluidic channel, and are allocated and redirected among a number ofparallel channels, wherein each parallel channel contains a portion ofeach of the samples and reflects the serial orientation originallypresented (bottom). Thus, serial to parallel conversion is alsoapplicable to performing fluidic operations which require large numbersof iterative or successive fluid manipulations, i.e., as in highthroughput analysis of samples where a plurality of different samples(e.g., 701, 702, 703 and 704) are subjected to a plurality of different:analyses (e.g., in each separate parallel channel). Specifically,separate channels each perform, in parallel, fluidic operations whichseparately require iterative and/or successive fluid manipulations.

While serial to parallel conversion is an important aspect of fluidcontrol in microfluidic systems, it does present difficulties from acontrol aspect. For example, fluid flow in electroosmotic systems iscontrolled by and therefore related to current flow between electrodes.Furthermore, resistance in the fluid channels varies as a function ofpath length and width, and thus, different length channels will havedifferent resistances. If this differential in resistance is notcorrected, it can result in the creation of transverse electrical fieldswhich can inhibit the ability of the devices to direct fluid flow toparticular regions within these devices. Specifically, the current, andthus the fluid flow will follow the path of least resistance, e.g., theshortest path. While this problem of transverse electrical fields isoptionally alleviated through the use of separate electrical systems,i.e., electrodes, at the termini of each and every parallel channel,production of devices incorporating all of these electrodes, and controlsystems for controlling the electrical potential applied at each ofthese electrodes are complex, particularly where one is dealing withhundreds to thousands of parallel channels in a single small scaledevice, e.g., 1-2 cm². Accordingly, the present invention providesmicrofluidic devices for affecting serial to parallel conversion, byensuring that current flow through each of a plurality of parallelchannels is at an appropriate level to ensure a desired flow patternthrough those channels or channel networks. FIGS. 8, 9 and 10 illustratea number of methods and substrate/channel designs for accomplishingthese goals.

In a first embodiment, FIG. 8 illustrates a substrate 800, employing achannel orientation that is optionally used to accomplish serial toparallel conversion or equal fluid flow in parallel channels. Thesubstrate includes main channel 802, which includes electrodes disposedin each of ports 804 and 806, at the termini of channel 802. A series ofparallel channels 808-822 and 830-844 terminate in main channel 802. Theopposite termini of these parallel channels are connected to parabolicchannels 824 and 846, respectively. Electrodes are disposed in ports826, 828, 848 and 850, which are included at the termini of theseparabolic channels, respectively.

In operation, a volume of fluid is transported along main channel 802 byapplying a potential across electrodes 804 and 806. An equal voltage isapplied across electrodes 826 and 828, and 848 and 850, resulting in anet zero flow through the parallel channels. The sample is optionallypresent within main channel 802 as a long slug of a single sample, ormultiple slugs of a single or multiple samples. Once the sample fluid orfluids reach the intersection of the main channel with the parallelchannels, e.g., 830-844, it is then pumped through the parallel channelsby applying a potential across electrode sets 826:828 and 848:850, whichresults in a fluid flow from parallel channels 808-822, to force thesamples into parallel channels 830-844. The current flow in each of theparallel channels 808-822 and 830-844 is maintained constant orequivalent, by adjusting the length of the parallel channels, resultingin a parabolic channel structure connecting each of the parallelchannels to its respective electrodes. The voltage drop within theparabolic channel between the parallel channels is maintained constantby adjusting the channel width to accommodate variations in the channelcurrent resulting from the parallel current paths created by theseparallel channels. For example, channel segment 824 a, while longer thanchannel segment 824 b, has the same resistance, because segment 824 a isappropriately wider. Thus, the parabolic design of channels 824 and 846,in combination with their tapering structures, results in the resistancealong all of the parallel channels being equal, resulting in an equalfluid flow, regardless of the path chosen. Generally, determining thedimensions of channels to ensure that the resistances among the channelsare controlled as desired, is optionally carried out by well knownmethods, and generally depends upon factors such as the make-up of thefluids being moved through the substrates.

In another example, FIG. 9 illustrates how the principles of the presentinvention can be used in a substrate design that employs fewerelectrodes to affect parallel fluid flow. In particular, fluid flowwithin an array of parallel channels is controlled by a single pair ofelectrodes. As shown, substrate 902 includes a plurality of parallelchannels 904-932. These parallel channels each terminate in transversechannels 934 and 936. Transverse channel 934 has a tapered width, goingfrom its widest at the point where it intersects the nearest parallelchannel 904 to the narrowest at the point it intersects the most distantparallel channel 932. Transverse channel 936, on the other hand, goesfrom its widest at the point it intersects parallel channel 932, to thenarrowest where it intersects parallel channel 902. Electrodes areincluded in the ports 938 and 940 at the wide termini of transversechannels 934 and 936, respectively. The dimensions of these taperedchannels are such that the current flow within each of the parallelchannels is equal, thereby permitting equal flow rates in each channel.As shown, transverse or sample introduction channel 942 is oriented sothat it crosses each parallel channel at the same point relative to oneor the other electrode, to ensure that the potential at theintersections of transverse channel 942 and all of the parallel channels904-932 is the same, again, to prevent the formation of transverseelectrical fields, or “shorting out” the array of channels. This resultsin the sample introduction channel 942 being disposed across theparallel channels at a non-perpendicular angle, as shown.

In operation, a sample fluid, e.g., disposed in port 944, is flowedthrough transverse channel 942, and across the intersection of theparallel channels 904-932 by applying a potential across ports 944 and946. Once the sample is disposed across the one or more desired parallelchannels, e.g., as dictated by the serial to parallel conversion desired(see, FIGS. 7A-7D), a potential is then applied across ports 938 and940, resulting in an equal fluid flow through each of the parallelchannels and injection of the sample fluid into each of the desiredparallel channels.

FIG. 10 illustrates still another embodiment for practicing theprinciples set forth herein. In this embodiment, a substrate includes alarge number of parallel channels. For ease of discussion, thesechannels are referred to herein as parallel channels 1004-1010, althoughit should be understood that preferred aspects will include upwards of20, 50, 100, 500 or more separate parallel channels. The parallelchannels 1004-1010 terminate at one end in transverse channel 1012 andat the other end in transverse channel 1014. Electrodes are providedwithin ports 1016 and 1018, and 1020 and 1022 at the termini of thesetransverse channels. In this embodiment, the problems of varying currentwithin the different parallel channels are addressed by providingtransverse channels 1012 and 1014 with sufficient width that voltagevariation across the length of these transverse channels, and thus, asapplied to each parallel channel, is negligible, or nonexistent.Alternatively, or additionally, a single electrode is optionallydisposed along the length of each of these transverse channels to ensureequal current flow at the transverse channel's intersection with eachparallel channel.

As shown, however, transverse or sample introduction channel 1024intersects each of the parallel channels, and is controlled byelectrodes disposed within ports 1026 and 1028 at the termini of channel1024. As described for FIG. 9, above, the sample introduction channelintersects each parallel channel at a point where the potential appliedto each channel will be equal. In this aspect, however, the channel isarranged substantially parallel to transverse channels 1012 and 1014, aseach parallel channel is subjected to the same voltages.

In operation, a sample, e.g., disposed in port 1026, is flowed throughsample channel 1024, across the intersection of the various parallelchannels 1004-1010, by applying a potential across ports 1026 and 1028.Once the sample fluid is in its appropriate location, i.e., across allor a select number of parallel channels, a potential is applied acrossports 1016:1020 and 1018:1022, injecting a plug of sample into theparallel channels.

The efficacy of these serial to parallel conversions was tested. Inbrief, a solid slug of fluorescent fluid material, e.g., includingfluorescein, rhodamine or the like, was injected through the diagonaltransverse channel by applying a potential across the transversechannel, e.g., at electrodes 944 and 946, such that the sample fluidspanned several of the parallel channels. By applying a potential acrossthe parallel channels, e.g., at electrodes 938 and 940, that portion ofthe fluid region at the intersections of the transverse channel and eachof the parallel channels was pumped down the parallel channels. Thesample fluid regions in each of the parallel channels was observed toflow at the same rate.

Parallel Fluid Manipulations

As described, the microfluidic systems of the present invention are alsoparticularly useful in performing fluidic operations that require alarge number of parallel fluid manipulations. Preferred systems canhandle processing of raw sample components through analysis of samplenucleic acids. This includes processing of biological samples such asblood such that DNA is available for analysis, providing an autosamplingsystem that can access external reagents or samples and import them foruse with the microchip processing components, and provide assays on themicrofluidic apparatus.

Two assays in the ultrahigh throughput format are particularlycontemplated: (1) size measurement for microsatellite typing of on-chipamplified DNA, and single nucleotide polymorphism (SNP) genotyping ofon-chip amplified DNA. In a particularly preferred aspect, these assaysare run using parallel microfluidics to maximize sample processingpower.

Sample Diagnostics

One example of a fluid operation that would benefit from the ability toperform rapidly large numbers of parallel manipulations is the screeningof a given sample in a number of separate assays. For example a singlefluid sample from a patient, e.g., blood, serum, saliva or the like, isscreened against a number of separate antibodies or antigens fordiagnostic testing. In a microfluidic format, this typically involvesthe apportioning of a single larger sample volume into numerous separateassay channels or chambers, wherein each separate chamber or channelcontains reagents for performing a different diagnostic assay. Forexample, in antibody panel screens, each reaction chamber or channel cancontain a different antibody or antigen. Such assay systems includethose described in U.S. patent application Ser. No. 08/671,987, filedJun. 28, 1996, and previously incorporated herein by reference.

Genotyping

Genetic analysis generally involves the correlation of measurablephysical traits (the phenotype) with the inheritance of particularversions of genetic elements like genotype). Genotyping of nucleic acidsamples from a patient typically involves a two step process. Because ofthe complexity of genomic information, the first step usually involvesan operation for reducing the complexity of the sample, or reducing thenumber of molecules in a mixture to be analyzed, into smaller but usefulportions. Once the complexity of the sample is reduced, the less complexsample is then optionally assayed for a particular genotype, or “typed.”This typing can be repeated upon a number of different segments or“loci” from the overall nucleic acid sample.

Reduction of sample complexity is typically carried out by biochemicalmethods that take a subset of the overall sample and concentrate itrelative to, or purify it away from the remainder of the sample.Examples of these biochemical methods include, e.g., amplifying aspecific subset of sample nucleic acids using preselected primers thatflank the desired segment. Alternatively, the desired segment is pulledfrom the larger sample by hybridization with a predefined probe that iscomplementary to all or a portion of the desired segment.

Once a nucleic acid sample is pared down to a manageable complexity, thesample is typed to identify the presence or absence of a particularvariation. Examples of such variations include simple sequence repeats(“SSR”), single nucleotide polymorphism (“SNP”), and small insertions ordeletions. In the case of SSRs, typing typically involves adetermination of the size of the sample segment, e.g., using size-basedelectrophoretic methods (gel exclusion), which will indicate thepresence or absence of a larger species corresponding to the samplesegment with or without the additional sequence elements. For SNPs andsmaller insertions or deletions, typing can be carried out by sequencingof the sample segment, to identify the base substitution, addition ordeletion. Such sequencing can be carried out by traditional sequencingmethods or by hybridization of the target sequence to oligonucleotidearrays, e.g., as described in U.S. Pat. No. 5,445,934, which is herebyincorporated herein by reference. Alternatively, the SNP or smallerinsertion or deletion can be identified by nuclease digestion of thesegment followed by size-based separation of the portions of thedigested segment. The pattern of fragments is then correlated with thepresence or absence of a particular marker sequence.

Typically, methods currently utilized in the art in these genotypingexperiments analyze each of the various different loci of the overallsample in a serial format. Specifically, the sample nucleic acid isamplified and characterized at a first locus, then at a second locus andso on. Further, such methods also typically utilize equipment that isonly capable of performing a single component of the overall process,e.g., amplification, electrophoresis, sequencing, etc. As set forthabove, the costs in equipment, time and space for performing thesemethods can be quite high, and increases substantially when a largenumber of samples and/or genetic loci are being screened.

According to the present invention, several if not all of the componentsof the overall process are integrated into a single microfluidic device.Further, multiple samples or disparate genetic loci from a single sampleare analyzed within a single device, in a parallel orientation. Forexample, because of the miniature format of the microfluidic devices,from about 1 to about 500 different genetic loci from a single nucleicacid sample can be analyzed in parallel, within a single device.

An example of a device for carrying out analysis of multiple loci on asingle nucleic acid sample is shown in FIG. 11. As shown, the device1100, is fabricated in a solid substrate 1102. The device includes amain sample channel 1114 which is intersected by multiple parallelseparation channels 1106-1118. Again, the number of these separationchannels on a single device can vary depending upon the desired size ofthe device. As shown, each of parallel separation channels 1106-1118 isfurther intersected by reagent introduction channels 1120-1132,respectively, and includes reaction chambers 1134-1146, respectively.The reagent introduction channels 1120-1132 have at their termini,reservoirs 1148:1150, 1152:1154, 1156:1158, 1160:1162, 1164:1166,1168:1170, and 1172:1174, respectively. Separation channels 1106-1118have at their termini opposite the sample introduction channel 1104,reservoirs 1176-1188 for applying a voltage across the separationchannel.

In operation, a fluid sample introduced into the sample introductionchannel 1104 is aliquoted among the separate parallel separationchannels 1106-1118 and delivered to reaction chambers 1134-1146,respectively. The sample is then treated according to the desiredprotocols by introducing into the reaction chambers reagents from thereservoirs at the termini of the reagent introduction channels.Following amplification, the target sequences are subjected to sizebased separation and analysis by transporting the amplified nucleicacids through the separation channels 1106-1118. Where the amplifiedsequence has a size that is different from the expected size of a“normal” individual it is indicative that sequence includes a sequencevariation, i.e., SSR. Alternatively, the amplified sequence is sequencedby well known sequencing methods. Such sequencing methods are optionallyincorporated into the devices described herein. For example, sequencingcan be carried out by the Sanger method by utilizing four of thereaction chambers for incorporation of each of the four ddNTPs.

Alternative substrate designs can also be used to accomplish the goalsof the device shown. In particular, as described in reference to serialto parallel conversion, above, a single reagent addition channel can beprovided which intersects all of the parallel separation channels.Reagents are then serially introduced into this main reagentintroduction channel and delivered to the various separation channelsand reaction chambers, using the serial to parallel conversion aspectsdescribed herein. Similarly, instead of providing a separate wastereservoir for each of the separation channels, a single transversechannel is optionally provided intersecting the separation channels attheir termini opposite the sample introduction channel. This singlechannel can be used to drive fluid flow, e.g., by applying a voltage atthe termini of this transverse channel. By reducing the number of portsat which voltage must be controlled, device design and control aresimplified, also as described herein.

Movement of Materials in Microscale Devices

As noted, the present invention provides microfluidic systems andmethods of using such systems in the performance of a wide variety offluidic operations and fluid manipulations. Microfluidic devices or“microlaboratory systems,” allow for integration of the elementsrequired for performing these operations or manipulations, automation,and minimal environmental effects on the reaction system, e.g.,evaporation, contamination, human error.

The phrase “selective direction” or “selective control” generally refersto the ability to direct or move a particular fluid volume from one areain a microfluidic device, e.g., a chamber or channel, to another area ofthe microfluidic device. Thus, selective direction includes the abilityto move one of several fluids contained within separate regions of amicrofluidic device without disturbing the other fluids, the directionof a portion of a fluid volume, as well as the ability to transport ordeliver an amount of a particular fluid from a first chamber to aselected one of several interconnected chambers.

Selective flowing, movement and direction of fluids within themicroscope fluidic devices is carried out by a variety of methods. Forexample, the devices optionally include integrated microfluidicstructures, such as micropumps and microvalves, or external elements,e.g., pumps and switching valves, for the pumping and direction of thevarious fluids through the device. Examples of microfluidic structuresare described in, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, 5,171,132,and 5,375,979. See also, Published U.K. Patent Application No. 2 248 891and Published European Patent Application No. 568 902.

Although microfabricated fluid pumping and valving systems are readilyemployed in the devices of the invention, the cost and complexityassociated with their manufacture and operation can generally prohibittheir use in mass-produced and potentially disposable devices as areenvisioned by the present invention. The devices of the invention willtypically include an electroosmotic fluid direction system. Such fluiddirection systems combine the elegance of a fluid direction systemdevoid of moving parts, with an ease of manufacturing, fluid control anddisposability. Examples of particularly preferred electroosmotic fluiddirection systems include, e.g., those described in International PatentApplication No. WO 96/04547 to Ramsey et al., as well as U.S. Ser. No.08/761,575 U.S. Pat. No. 6,046,056 by Parce et al. and U.S. Ser. No.08/845,754 now U.S. Pat. No. 5,976,336 to Dubrow et al.

In brief, these fluidic control systems typically include electrodesdisposed within reservoirs that are placed in fluid connection with -thechannels fabricated into the surface of the substrate. The materialsstored in the reservoirs are transported through the channel systemdelivering appropriate volumes of the various materials to one or moreregions on the substrate in order to carry out a desired screeningassay.

Material transport and direction is accomplished through electrokinesis,e.g., electroosmosis or electrophoresis. In brief, when an appropriatefluid is placed in a channel or other fluid conduit having functionalgroups present at the surface, those groups can ionize. For example,where the surface of the channel includes hydroxyl functional groups atthe surface, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface will possess a net negativecharge, whereas the fluid will possess an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid. By applying an electric field along the length ofthe channel, cations will flow toward the negative electrode. Movementof the positively charged species in the fluid pulls the solvent withthem. An electrokinetic device moves components by applying an electricfield to the components, typically in a microfluidic channel. Byapplying an electric field along the length of the channel, cations willflow toward a negative electrode, while anions will flow towards apositive electrode. Movement of the charged species in the fluid pullsthe solvent with the fluid. The steady state velocity of this fluidmovement is generally given by the equation:$v = \frac{\varepsilon \quad \xi \quad E}{4\quad \pi \quad \eta}$

where v is the solvent velocity, ε is the dielectric constant of thefluid, ξ is the zeta potential of the surface, E is the electric fieldstrength, and η is the solvent viscosity. The solvent velocity is,therefore, directly proportional to the surface potential.

To provide appropriate electric fields, the system generally includes avoltage controller that is capable of applying selectable voltagelevels, simultaneously, to each of the reservoirs, including ground.Such a voltage controller can be implemented using multiple voltagedividers and multiple relays to obtain the selectable voltage levels.Alternatively, multiple, independent voltage sources are used. Thevoltage controller is electrically connected to each of the reservoirsvia an electrode positioned or fabricated within each of the pluralityof reservoirs. In one embodiment, multiple electrodes are positioned toprovide for switching of the electric field direction in a microchannel,thereby causing the analytes to travel a longer distance than thephysical length of the microchannel.

Substrate materials are also selected to produce channels having adesired surface charge. In the case of glass substrates, the etchedchannels will possess a net negative charge resulting from the ionizedhydroxyls naturally present at the surface. Alternatively, surfacemodifications are employed to provide an appropriate surface charge,e.g., coatings, derivatization, e.g., silanation, or impregnation of thesurface to provide appropriately charged groups on the surface. Examplesof such treatments are described in, e.g., Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 Ser. No.08/843,212 filed Apr. 14, 1997, now U.S. Pat. No. 5,885,470.

Modulating voltages are then concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous or discontinuous (e.g., a regularly pulsed field causing theflow to oscillate direction of travel) flow of receptor/enzyme,ligand/substrate toward the waste reservoir with the periodicintroduction of test compounds. Particularly, modulation of the voltagesapplied at the various reservoirs can move and direct fluid flow throughthe interconnected channel structure of the device in a controlledmaimer to effect the fluid flow for the desired screening assay andapparatus.

While a number of devices for carrying out particular methods accordingto the invention are described in substantial detail herein, it will berecognized that the specific configuration of these devices willgenerally vary depending upon the type of manipulation or reaction to beperformed. The small scale, integratability and self-contained nature ofthese devices allows for virtually any reaction orientation to berealized within the context of the microlaboratory system.

Because the microfluidic devices of the invention preferably employelectroosmotic fluid direction systems, and are substantially sealed tothe outside environment, excepting reagent, buffer or sample ports, theyare capable of performing fluidic operations while maintaining precisecontrol of the amounts of different fluids to be delivered to thedifferent regions of the substrate.

For example, the sealed nature of the devices prevents substantialevaporation of fluids from the devices. Evaporation, while a problem atthe bench scale, becomes substantially more problematic when operatingat the microscale, where loss of minute amounts of fluids can have adramatic effect on concentrations of the non volatile elements of thesefluids, particularly where extended reaction times are concerned. Thus,the devices and systems of the invention provide the added advantage ofperforming fluidic operations with a controlled volume. By “controlledvolume” is meant that the systems can transport or direct a particularvolume of a particular fluid which is generally within about 10% of anexpected or desired volume or amount of that fluid, preferably withinabout 5% of an expected or desired volume, and often within about 1% ofan expected or desired volume.

The phrase “preselected volume” or simply “selected volume” refers to avolume of fluid that is to be subjected to a particular fluidmanipulation. Again, as noted above, in the fluid filled chambers,channels and/or reservoirs of the systems of the invention, thesepreselected volumes are generally transported as slugs of differentfluids within these fluid filled elements. Generally, a preselectedvolume will be within at least about 10% of a desired volume. Thus,where one wishes to transport a preselected volume of 1 μl of aparticular fluid from a first chamber to a second chamber, the fluiddirection systems of the present invention would transport 1 μl±10%. Inpreferred aspects, these systems will maintain a volume within about 5%and often, within about 1%. In addition to reliable volumetric control,the fluid direction systems of the present invention are generallycapable of moving or directing small preselected fluid volumes. Forexample, the fluid direction systems of the present invention aregenerally capable of selectively directing volumes of fluid that areless than about 10 μl, preferably less than about 1 μl, more preferablyless than 0.1 μl and often less than about 10 nl.

In addition to the volume advantages discussed above, the sealed natureand readily automatable fluid direction systems also protects fluidoperation performer in these devices from contaminating influences fromthe outside environment. Such influences include chemical, biological ormicrobiological contamination of fluidic operations which can affect anoutcome of such operations. In addition, such contaminating influencescan include the occurrence of human error that is generally associatedwith manual operations, e.g., measurement errors, incorrect reagentadditions, detection errors and the like.

High quality data generation is achieved through two basic levels ofcontrol: “hardware-level” control whereby the instruction set forperforming a fluidic operation experiment is coded in fine channels(e.g., 10-100 μm wide, 1-50 μm deep), and “software-level” controlwhereby the movement of fluid and/or materials through the channelnetwork is controlled with exquisite precision by manipulating electricfields introduced into the network through electrodes at channel terminiusing the methods discussed above. Integrated volumetrics capable ofhighly precise, sub-nanoliter measurements and dispensing are a featureof this invention. Electronics that allow simultaneous,millisecond-resolution control over large voltage gradients or currentchanges disposed across the different parts of complex LabChipstructures are performed using the techniques described above. Thispermits fluid or material flow at intersections to be accuratelycontrolled and providing “virtual valves”, structures that meter fluidby electronic control with no moving parts. The electric field controland small conduit dimensions allow experimentation to be performed onsub-nanoliter fluid volumes.

Detectors

The substrate typically includes a detection window or zone at which asignal is monitored. This detection window typically includes atransparent cover allowing visual or optical observation and detectionof the assay results, e.g., observation of a colorometric, fluorometricor radioactive response, or a change in the velocity of colorometric,fluorometric or radioactive component. Detectors often detect a labeledcompound, with typical labels including fluorographic, colorometric andradioactive components. Example detectors include spectrophotometers,photodiodes, microscopes, scintillation counters, cameras, film and thelike, as well as combinations thereof. Examples of suitable detectorsare widely available from a variety of commercial sources known topersons of skill.

In one aspect, monitoring of the signals at the detection window isachieved using an optical detection system. For example, fluorescencebased signals are typically monitored using, e.g., in laser activatedfluorescence detection systems which employ a laser light source at anappropriate wavelength for activating the fluorescent indicator withinthe system. Fluorescence is then detected using an appropriate detectorselement, e.g., a photomultiplier tube (PMT). Similarly, for screensemploying colorometric signals, spectrophotometric detection systems areemployed which detect a light source at the sample and provide ameasurement of absorbance or transmissivity of the sample. See also, ThePhotonics Design and Applications Handbook, books 1, 2, 3 and 4,published annually by Laurin Publishing Co., Berkshire Common, P.O. Box1146, Pittsfield, Mass. for common sources for optical components.

In alternative aspects, the detection system comprises non-opticaldetectors or sensors for detecting a particular characteristic of thesystem disposed within detection window 116. Such sensors optionallyinclude temperature (useful, e.g., when a reaction produces or absorbsheat, or when the reaction involves cycles of heat as in PCR or LCR),conductivity, potentiometric (pH, ions), amperometric (for compoundsthat can be oxidized or reduced, e.g., O₂, H₂O₂, I₂,oxidizable/reducible organic compounds, and the like).

Alternatively, schemes similar to those employed for the enzymaticsystem are optionally employed, where there is a signal that reflectsthe interaction of the receptor with its ligand. For example, pHindicators which indicate pH effects of receptor-ligand binding can beincorporated into the device along with the biochemical system, i.e., inthe form of encapsulated cells, whereby slight pH changes resulting frombinding can be detected. See Weaver, et al., Bio/Technolog (1988)6:1084-1089. Additionally, one can monitor activation of enzymesresulting from receptor ligand binding, e.g., activation of kinases, ordetect conformational changes in such enzymes upon activation, e.g.,through incorporation of a fluorophore which is activated or quenched bythe conformational change to the enzyme upon activation.

One conventional system carries light from a specimen field to a cooledcharge-coupled device (CCD) camera. A CCD camera includes an array ofpicture elements (pixels). The light from the specimen is imaged on theCCD. Particular pixels corresponding to regions of the substrate aresampled to obtain light intensity readings for each position. Multiplepositions are processed in parallel and the time required for inquiringas to the intensity of light from each position is reduced. Thisapproach is particularly well suited to DNA sequencing, because DNAsequencing products are easily labeled using any of a variety offluorophores known in the art. Many other suitable detection systems areknown to one of skill.

Computers

Data obtained (and, optionally, recorded) by the detection device istypically processed, e.g., by digitizing the image and storing andanalyzing the image on a computer readable medium. A variety ofcommercially available peripheral equipment and software is availablefor digitizing, storing and analyzing a signal or image. A computer iscommonly used to transform signals from the detection device intosequence information, reaction rates, or the like. PC (Intel x86 orpentium chip- compatible DOS™, OS2™ WINDOWS ™ WINDOWS NT™, WINDOWS95™ orWINDOWS97™ based machines), MACINTOSH™, or UNIX™ based (e.g., SUN™ workstation) computers are all commercially common, and known to one ofskill. Software for determining reaction rates or monitoring formationof products, or for translating raw sizing data for sequencing productsinto actual sequence are available, or can easily be constructed by oneof skill using a standard programming language such as Visualbasic,Fortran, Basic, Java, or the like. The software is optionally designedto determine product velocities, concentrations, flux relationships,sequence information and the like as described, supra. Any controller orcomputer optionally includes a monitor which is often a cathode ray tube(“CRT”) display, a flat panel display (e.g., active matrix liquidcrystal display, liquid crystal display), or others. Computer circuitryis often placed in a box which includes numerous integrated circuitchips, such as a microprocessor, memory, interface circuits, and others.The box also optionally includes a hard disk drive, a floppy disk drive,a high capacity removable drive (e.g., ZipDrive™ sold by lomegaCorporation), and other elements. Inputing devices such as a keyboard ormouse optionally provide for input from a person.

More generally, the microfluidic systems herein typically includecontrol systems for carrying out one or more operations of: controllingfluid movement and direction; monitoring and controlling environmentaleffects on a microfluidic device; and recording and analyzing dataobtained from the microfluidic devices. Typically, such control systemsinclude a programmable computer or processor that is linked, via anappropriate interface, with the other elements of the system. Forexample, the computer or processor will typically interface with: thevoltage controller, to direct the electroosmotic fluid direction system;with a detector disposed adjacent the detection window, to obtain datafrom the device; and with the device itself, to maintain appropriatereaction conditions within the device, e.g., temperature.

Computerized control of the microfluidic systems allows for therepeated, automatic and accurate performance of the various fluidicoperations performed within a microfluidic device, or within severaldevices, simultaneously. Further, the computer is generally programmableso that a user can modify protocols and/or conditions as desired, aswell as to record, compile and analyze the data from the device, e.g.,statistical analysis. A block diagram of a control system as connectedto a microfluidic device is shown in FIG. 12. In particular, the overallsystem 1200 includes a microfluidic device 1202, a voltage controller1204, a detector 1206, and a computer or processor 1208. The voltagecontroller is connected to electrodes 1210-1216 which are placed inelectrical contact with fluids in the various ports of the microfluidicdevice 1202. The voltage controller is, in turn connected to computer1208. This connection can also include an appropriate AD/DA converter.The computer 1208 is also connected to detector 1206, for instructingoperation of the detector, as well as recording data obtained by thedetector. Detector 1206 is typically disposed adjacent to an appropriatedetection window 1220 within the microfluidic device. In alternateaspects, a detector can be incorporated within the device itself.

Integrated Systems e.g. for Sequencing, Thermocycling, AssayOptimization and Drug Screening

The present invention is further illustrated by consideration of theaccompanying figures.

FIG. 13 provides an embodiment of the invention having anelectropipettor integrated into a microfluidic substrate having a fluidmixing region, a thermocycler region, a size separation region and adetection region. In operation, reagent storage substrate 1300 havingdried reagent dots, e.g., 1320-1330 is suspended above or belowmicrofluidic substrate 1305 having channels 1345-1355, intersecting atchannel intersections 1360 and 1365 and reagent wells 1370-1390.Channels 1345, 1350 and 1355 are fluidly connected to electropipettor1395 and channel 13100 in electropipettor 1395. As depicted, optionalreagent mixing chamber 13103 provides for mixing of reagents fromsubstrate 1310 prior to entry into channels 1345-1355. In oneembodiment, enzyme for a sequencing reaction (i.e., a polymerase enzyme)is stored in well 1390, while dNTPs are stored in well 1385; thecomponents are mixed e.g., in channel 1365 or channel 1345. This isuseful, e.g., in embodiments where modular primers are used in asequencing reaction and more than one primer is needed for thesequencing reaction. It will be appreciated that chamber 13103 isoptionally omitted, in which case electropipettor channel 13100 andsubstrate channels 1345-1355 are directly connected.

Electropipettor tip 13105 is fitted to expel fluid onto primer dots1315-1330 and to then draw the resulting solubilized primer into channel13100 for further processing in channels 1345-55, which optionallyinclude mixing, heating, or cooling portions. Reaction products areseparated in channel 1340 having detection zone 13110. Products movingthrough detection zone 13110 are detected by detector 13115 operablycoupled to computer 13120. In sequencing embodiments, reagent storagesubstrate 1310 typically has most or all of the possible primers of agiven length, e.g., 4,096 6-mer primers, e.g., in 4,096 separate dots(optionally more than one primer can exist in a single dot, with theselection of sequencing primers taking all of the primers in each dotinto account as compared to the template nucleic acid). Computer 13120is used to select extension primers from reagent storage substrate 1310according to the selection methods described herein. Sequencingreactions are carried out in channels 1345-1365, optionally includingPCR in selected sections of the channels. Sequencing products aredetected by detector 13115, and the detection is converted intosequencing information in computer 13120.

Although depicted with reagent storage substrate 1310 over microfluidicsubstrate 1335, it will be appreciated that reagent storage substrate1310 can conveniently be either above or below microfluidic substrate1335. In addition, although depicted with dried reagents, reagentstorage substrate 1310 can be substituted with a microtilter dish havingreagents in liquid form, although a microtiter dish will usually belocated below microfluidic substrate 1335.

FIG. 14 depicts an alternate embodiment to FIG. 13, in whichelectropipettor 1405 is in the same plane as microfluidic substrate1410. Channels 1415, 14140 and 1430 in substrate 1410 are fluidlyconnected to wells 1435, 1440 and 1445, and are also fluidly connectedto channel 1450 in electropipettor 1405 through optional mixing chamber1407 As depicted, optional reagent mixing chamber 1407 provides formixing of reagents from substrate 1455 prior to entry into channels1415, 1420 and 1430. This is useful, e.g., in embodiments where modularprimers are used in a sequencing reaction and more than one primer isneeded for the sequencing reaction. It will be appreciated that chamber1407 is optionally omitted, in which case electropipettor channel 1450and substrate channels 1415, 1420 and 1430 are directly connected.Reagent storage substrate 1455 having dried reagent dots such as dot1460 is perpendicular (or at an angle) to substrate 1410.Electropipettor 1405 solubilizes dots on reagent storage substrate 1455by expelling liquid from electropipettor tip 1470 onto dots such as dot1460, thereby solubilizing the reagent(s) in dot 1460, and withdrawingthe reagent(s) into electropipettor tip 1470, channel 1450 andsubsequently into substrate 1410. After mixing with additional reagents,e.g. stored in wells 1435, or 1445 and any resulting reaction, reactionproducts are incubated and separated in channel 1420 and detected indetection region 1475 by detector 1480. Waste materials are stored,e.g.,, in well 1440. Information regarding the detection is digitizedand fed into operably linked computer 1485. As discussed above, thecomputer translates the information into, e.g., sequence information,drug discovery information or the like and directs selection of a secondreagent dot on substrate 1455 (e.g., a second primer) for analysis.

In the embodiments depicted in FIG. 13 and FIG. 14, computers 1320 and1485 typically store information regarding the location of reagent dotson reagent storage substrates 1310 and 1455. Typically this will be inthe form of address information, where the address of each reagent doton regent storage substrates 1310 or 1455 is stored for subsequentselection steps. Either the relevant microfluidic substrate,electropipettor or reagent storage substrate is moved so that theelectropipettor contacts the selected reagent dot (any or all of thecomponents can be moved to cause the electropipettor to contact theproper point on the particular reagent storage substrate. Movement canbe conveniently achieved using a mechanical armature in contact with thecomponent to be moved. Alternatively, the components can be movedmanually.

FIG. 15 is an alternate preferred embodiment in which electropipettorchannel 1510 is contiguous with microfluidic channels 1520 1530 and 1540which are connected to wells 1550, 1560 and 1570, respectively. In thisembodiment, microfluidic substrate 1580 comprises electropipettor tip1590, which includes electropipettor channel 1510.

FIG. 16 is an additional alternate preferred embodiment in whichelectropipettor capillary 1610 protrudes from microfluidic substrate1620. Capillary 1610 is in fluid communication with microfluidic channel1625, which is in fluid communication with channels 1635, 1640, and 1645and wells 1650-1680.

FIG. 17 is an additional preferred embodiment similar to that depictedin FIG. 15. In operation, electropipettor channel 1710 inelectropipettor tip 1720 is fluidly connected to microfluidic channels1725-1755 and wells 1760-1797 in microfluidic substrate 1799.

It will be appreciated that the embodiments depicted in FIGS. 15-17 caneasily by used in an integrated apparatus similar to that depicted inFIG. 13 or FIG. 14, i.e., comprising a reagent storage substrate,armature for moving the reagent substrate and/or the microfluidicsubstrate, a viewing apparatus such as a microscope or photodiode and acomputer for processing data, controlling fluid movement on thesubstrate, and controlling movement of electropipettor componentsrelative to the reagent storage substrate.

Furthermore, it will be appreciated that a variety of reagent storagesubstrates are appropriate. For example, FIG. 18 provides a preferredintegrated apparatus in which reagents to be selected are stored inliquid form. In operation, liquid reagents are stored in liquid reagentstorage tray 1805. The reagents are stored in wells 1810 located inreagent storage tray 1805. A variety of reagent storage trayscommercially available are suitable for this purpose, includingmicrotiter dishes, which are available e.g. in a 918-well format.Microfluidic substrate 1815 is located over reagent storage tray 1805.For convenience of manipulation, either microfluidic substrate 1815 ormicrotiter tray 1805 or both can be moved using a robotic armature. Asdepicted, robotic movable armature 1815 is connected to microfluidicsubstrate 1810 and moves the substrate relative to reagent storage tray1805 in response to instructions from computer 1820. Similarly, roboticmovable armature 1825 is attached to and moves reagent storage tray 1805relative to microfluidic substrate 1810 in response to instructions fromcomputer 1820. It will be appreciated that to move microfluidicsubstrate 1810 relative to reagent storage tray 1805, only one movablearmature is need; accordingly, either armature 1815 or armature 1825 isoptionally omitted. Similarly, either armature 1815 or armature 1825 canbe replaced with a movable platform or the like for moving microfluidicsubstrate 1810 relative to reagent storage tray 1805, or vice versa.

In operation, microfluidic substrate 1810 is sampled by electropipettor1830 for sampling reagents from wells 1810. Electropipettor 1830 isfluidly connected to microchannels 1835-1850 and microfluidic substratewells 1860-18100. Reagent mixing, electrophoresis and the like isperformed in microchannels 1835-1850. Typically, an electrokineticcontrol apparatus such as voltage controller connected to electrodeslocated in one or more of microfluidic substrate wells 1860-700 controlsmaterial transport through microchannels 1835-1850. Detector 18110detects the results of fluidic mixing assays, such as fluorescentsequencing products, inhibition assays, titrations or the like. Theresults detected are digitized and read by computer 1820, which selectsadditional fluidic reagents for additional assays, based upon theresults detected. Selection of additional reagents causes movement ofmovable robotic armature 1825 or 1815, thereby positioningelectropipettor 1830 in well 1810 having the selected reagent.

FIG. 19 is an outline of the computer processing steps typical indetermining sequence information and in selecting primers useful in themethods and apparatus described herein. Additional processing stepsperformed to run a voltage controller to direct fluid movement in amicrofluidic substrate are optionally performed by the computer.

FIG. 20 provides an embodiment of the invention directed to sequencing.Template DNAs (e.g., single-stranded cosmid DNA, plasmid DNA, viral DNAor the like) to be sequenced is stored in well 2010. The template DNAsare conveniently complexed with capture beads. Sequencing reagents(polymerase, dNTPs, ddNTPs or the like) are stored in well 2015. Buffersfor material transport, and or reagents are stored in wells 2020-2030.Electropipettor channel 2035 is connected to a source of all possible6-mer primers, as described, supra. Template DNA on capture beads (e.g.,posts, magnitic beads, polymer beads or the like) from well 2010 iselectrokinetically transported to bead capture area 2040. Appropriateprimers are selected and transported to bead capture 2040 area usingelectropipettor channel 2035. Polymerase from well 2015 is contactedwith to template DNA in bead capture area 2040. Extension of primers onthe template with the polymerase results in sequencing products. Theproducts are washed from the template using loading buffer from well2020 (the loading buffer optionally comprises a denaturant) andelectrophoresed through size separation microchannel 2043. The productsseparate by size, permitting detection of the products with detector2045, which is operatively linked to computer 2050. After detection,products enter waste well 2055. After size detection and analysis,computer 2050 directs selection of additional primers to extendsequencing of the template DNAs. Once all of the template is sequencedby repeated cycles of sequencing, the template and beads are in optionalembodiments released from bead capture area 2040 using buffer from well2030 or 2025 and the template DNA beads are transported to waste well2060. Additional templates are then loaded into well 2010 and theprocess is repeated with the additional templates.

The labChip depicted in FIG. 21 was used to perform multiple operationsin a biochemical assay were run on the chip. This demonstrates theability to integrate functions such as complex (blood) samplepreparation, specialized reaction (polymerase chain reaction, PCR), andsophisticated analysis (DNA size separation) in a single format.

In the experiment, LabChip™ 2110 was used to prepare wole blood, loadDNA template from whole blood, run the PCR reaction and then size theresulting PCR product by gel separation. Channels 2130 and 2140 werefilled with sieving matrix gel 2150. In addition, wells 2160 and 2170 atthe ends of separation channel 2130 were filled with gel. For the firstpart of the experiment, approximately 2000 lymphocytes (white bloodcells) purified from whole blood in a conventional way (centrifugation)were added to 20 μL of PCR reaction mix and placed in sample well 2180of chip 2110. The wells were overlaid with mineral oil and the chip wascycled using a thermocycler. After cycling, the PCR product wasseparated by passage through a second chip through channel 2130. FIG. 22shows the electropherogram for this portion where the amplified peak ofthe HLA locus (about 300 bp) is seen at around 34 seconds at the sametime as the 270-310 bp fragments in the PhiX 174 standard ladder. Forthe second part of the experiment, PCR reaction mix without DNA templatewas placed in well 2180 of a fresh chip and 5% whole blood in which thered blood cells had been lysed was placed in another well. Lymphocytes(white blood cells) were electrophoresed through the channel to the wellcontaining the PCR reaction mixture until 20-100 lymphocytes were in thePCR well. The chip was cycled and DNA separated as for the previouschip. The results are shown in FIG. 23. Amplification was achieved forboth purified and electrophoresed lymphocytes, although the amount ofproduct for purified lymphocytes was larger than for electrophoresedlymphocytes. Sufficient PCR cycles were run to ensure that the reactionhad reached a plateau stage since the number of starting copies; wasdifferent. These experiments demonstrate the ability to integrateseveral steps of a complex biochemical assay on a microchip format.

Modifications can be made to the method and apparatus as hereinbeforedescribed without departing from the spirit or scope of the invention asclaimed, and the invention can be put to a number of different uses,including:

The use of an integrated microfluidic system to test the effect of eachof a plurality of test compounds in a biochemical system in an iterativeprocess.

The use of an integrated microfluidic system as hereinbefore described,wherein said biochemical system flows through one of said channelssubstantially continuously, enabling sequential testing of saidplurality of test compounds.

The use of a microfluidic system as hereinbefore described, wherein theprovision of a plurality of reaction channels in said first substrateenables parallel exposure of a plurality of test compounds to at leastone biochemical system.

The use of a microfluidic system as hereinbefore described, wherein eachtest compound is physically isolated from adjacent test compounds.

The use of a substrate carrying intersecting channels in screening testmaterials for effect on a biochemical system by flowing said testmaterials and biochemical system together using said channels.

The use of a substrate as hereinbefore described, wherein at least oneof said channels has at least one cross-sectional dimension of range 0.1to 500 μm.

The use of an integrated system as described herein for nucleic acidsequencing.

An assay, kit or system utilizing a use of any one of the microfluidiccomponents, methods or substrates hereinbefore described. Kits willoptionally additionally comprise instructions for performing assays orusing the devices herein, packaging materials, one or more containerswhich contain assay, device or system components, or the like.

In an additional aspect, the present invention provides kits embodyingthe methods and apparatus herein. Kits of the invention optionallycomprise one or more of the following: (1) an apparatus or apparatuscomponent as described herein; (2) instructions for practicing themethods described herein, and/or for operating the apparatus orapparatus components herein; (3) one or more assay component; (4) acontainer for holding apparatus or assay components, and, (5) packagingmaterials.

In a further aspect, the present invention provides for the use of anyapparatus, apparatus component or kit herein, for the practice of anymethod or assay herein, and/or for the use of any apparatus or kit topractice any assay or method herein.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications and patentdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication or patent document were so individually denoted.

What is claimed is:
 1. A method of sequencing a nucleic acid comprising:providing a target nucleic acid, a first sequencing primer, apolymerase, ddNTPs, and ddNTPs; mixing the target nucleic acid, thefirst sequencing primer, the polymerase, the dNTPs, acid the ddNTPs in amicrofluidic device under conditions permitting target dependentpolymerization of the dNTPs, thereby providing polymerization products;and, separating the polymerization products by size in the microfluidicdevice to provide a first sequence of the target nucleic acid.
 2. Themethod of claim 1, wherein a second sequencing primer is selected basedupon the sequence of the target nucleic acid and the second sequencingprimer is mixed with the target nucleic acid in a microfluidic deviceunder conditions permitting target dependent elongation of the selectedsecond sequencing primer thereby providing polymerization products whichare separated by size in the microfluidic device to provide a secondsequence of the target nucleic acid.
 3. The method of claim 1, whereinthe first sequencing primer is selected from a large set of sequencingprimers which comprises a primer having a sequence complementary to thetarget nucleic acid.
 4. The method of claim 3, wherein the large setcomprises at least about 70% of all possible sequencing primers for agiven length, wherein the length is between about 4 and about
 10. 5. Themethod of claim 3, wherein the large set comprises at least 3,000different oligonucleotide members.
 6. A method of sequencing a targetnucleic acid, comprising: (a) providing an integrated microfluidicsystem comprising a microfluidic device comprising: at least a firstsequencing reaction channel and at least a first sequencing reagentintroduction channel, the sequencing reaction channel and sequencingreagent introduction channel being in fluid communication; and, afluidic interface in fluid communication with the sequencing reagentintroduction channel for sampling a plurality of sequencing reagents ormixtures of sequencing reagents from a plurality of sources ofsequencing reagents or mixtures of sequencing reagents and introducingthe sequencing reagents or mixtures of sequencing reagents into thesequence reagent introduction channel from the sources of sequencingreagents or mixtures of sequencing reagents; selecting a firstsequencing primer sequence complementary to a first subsequence of afirst target nucleic acid sequence; (b) introducing the first sequencingprimer and the first target nucleic acid sequence into the sequencereagent introduction channel; (c) hybridizing the first primer sequenceto the first subsequence in the first sequencing reaction channel andpolymerase-extending the first primer sequence along the length of thetarget nucleic acid sequence to form a first extension product that iscomplementary to the first subsequence and a second subsequence of thetarget nucleic acid; (d) determining the sequence of the first extensionproduct; (e) based upon the sequence of the first extension product,selecting a second primer sequence complementary to the secondsubsequence of the target nucleic acid sequence; (f) hybridizing thesecond primer sequence to the second subsequence in the first sequencingreaction channel, or, optionally, hybridizing the second primer sequencein the second subsequence in a second sequencing reaction channel; (g)extending the second primer sequence along the length of the targetnucleic acid sequence to form a second extension product that iscomplementary to the second subsequence and a third subsequence of thetarget nucleic acid sequence; and (h) determining the sequence of thesecond extension product.
 7. The method of claim 6, wherein the secondprimer is selected using a computer.
 8. The method of claim 6, whereinthe microfluidic device comprises a material transport system forcontrollably transporting sequencing reagents through the sequencingreagent introduction channel and sequencing reaction channel.
 9. Amethod of sequencing a nucleic acid comprising: providing a set ofsequencing primers, a target nucleic acid, a polymerase, dNTPs, andddNTPs; selecting a first primer from the set of primers; introducingthe first primer into a microfluidic device; mixing the first primer,the polymerase, the dNTPs, and the ddNTPs in a first zone of themicrofluidic device under conditions permitting target dependentpolymerization of the dNTPs, thereby providing polymerization products;separating polymerization products by size in a second zone of themicrofluidic device to provide at least a first portion of the sequenceof the target nucleic acid; selecting a second primer from the set ofprimers, which primer is complementary to the first portion of thetarget nucleic acid; introducing the second primer into the microfluidicdevice; mixing the second primer, the polymerase, the dNTPs, and theddNTPs in a third zone of the microfluidic device under conditionspermitting target dependent polymerization of the dNTPs, therebyproviding polymerization products; separating polymerization products bysize in a fourth zone of the microfluidic device to provide at least asecond portion of the sequence of the target nucleic acid.
 10. Themethod of claim 9, wherein the first and third zone of the microfluidicdevice are the same and wherein the second and fourth zone of themicrofluidic device are the same.
 11. The method of claim 9, wherein theset of primers is located on the microfluidic apparatus.
 12. The methodof claim 9, wherein mixing the second primer, polymerase, dNTPs, ddNTPsin a microfluidic device under conditions permitting polymerization andseparating polymerization products by size to provide at least a portionof the sequence of the target nucleic acid is performed in less than 15minutes.
 13. The method of claim 9, further comprising selecting a thirdprimer from the set of primers, which third primer is complementary tothe second portion of the target nucleic acid; mixing the third primer,the polymerase, the dNTPs, and the ddNTPs in a microfluidic device underconditions permitting target dependent polymerization of the dNTPs,thereby providing polymerization products; separating polymerizationproducts by size to provide at least a third portion of the sequence ofthe target nucleic acid.
 14. An integrated method of performing afluidic analysis of sample materials, comprising: (a) providing anintegrated microfluidic system comprising a microfluidic devicecomprising: at least a first reaction chamber or channel, and at least afirst reagent introduction channel, the first reaction chamber orchannel and reagent introduction channel being in fluid communication; amaterial transport system for controllably transporting a materialthrough the reagent introduction channel and reaction chamber orchannel; a fluidic interface in fluid communication with the reagentintroduction channel for sampling a plurality of reagents or mixtures ofreagents from a plurality of sources of reagents or mixtures of reagentsand introducing the reagents or mixtures of reagents into the reagentintroduction channel; (b) selecting a first set of DNA sequencingreagents comprising a first nucleic acid sequencing primer from theplurality of sources of sequencing reagents or mixtures of sequencingreagents; (c) introducing a first DNA template and the first set of DNAsequencing reagents comprising a first nucleic acid sequencing primerinto the first reaction chamber or channel whereupon the first DNAtemplate and the first set of DNA sequencing reagents comprising a firstnucleic acid sequencing primer react to produce a first set of DNAsequencing products; (d) analyzing the first set of DNA sequencingproducts by separating the DNA sequencing products by size and detectingthe size separated DNA sequencing products, thereby providing sequenceinformation for the first DNA template; (e) based upon the the sequenceinformation determined in step (d), selecting a second set of DNAsequencing reagents comprising a second sequencing primer and a secondDNA template; (f) introducing the second set of DNA sequencing reagentscomprising a second sequencing primer and the first DNA template intothe first reaction chamber or channel, or, optionally, into a secondreaction chamber or channel in the microfluidic device, whereupon thesecond set of DNA sequencing reagents comprising a second sequencingprimer and the first DNA template react to produce a second set of DNAsequencing products; and (g) analyzing the second set of DNA sequencingproducts to provide additional sequence information for the first DNAtemplate.
 15. The method of claim 14, wherein the first or secondreagents or mixtures of reagents comprise a thermostable polymerase andthe method further comprises heating the first sample material and thefirst reagent or mixture of reagents or the second sample materials andthe second reagent or mixture of reagents.
 16. The method of claim 14,wherein the second sequencing primer is selected using a computer.
 17. Amethod of performing iterative fluid operations, comprising: providing amicrofluidic system which comprises: a microfluidic device having atleast a first microscale channel disposed therein, the at least firstchannel being in fluid communication with a plurality of reagentsources; a material transport system for transporting reagents from eachof the plurality of reagent sources into the at least first channel;introducing at least a first nucleic acid template from at least a firstreagent source and a first sequencing primer from at least a secondreagent source into the at least first channel, the nucleic acidtemplate and first sequencing primer producing a first elongationproduct; detecting a nucleotide sequence of the first elongationproduct; and based upon the reaction of the first and second reagents,selecting and introducing a an additional amount of the nucleic acidtemplate and a second sequencing primer into the at least first channelthe second sequencing primer based upon the nucleotide sequence of thefirst elongation product.
 18. The method of claim 17, wherein the secondsequencing primer comprises a sequence identical to at least a portionof the first elongation reaction product.
 19. The method of claim 17,wherein the at least first sequencing primer comprises a plurality ofnucleic acid primers that contiguously anneal to the nucleic acidtemplate.
 20. The method of claim 17, wherein the at least firstsequencing primer comprises at least 2 contiguously annealing primers.21. The method of claim 17, wherein the at least first sequencing primercomprises at least 3 contiguously annealing primers.
 22. The method ofclaim 19, wherein the plurality of contiguously annealing primers are 5or 6 nucleotides in length.
 23. The method of claim 19, wherein theplurality of contiguously annealing primers are independently selectedfrom a plurality of primer sources comprising all possible primers of 5or 6 nucleotides in length.
 24. The method of claim 17, wherein thesecond sequencing primer comprises a plurality of nucleic acid primersthat contiguously anneal to the nucleic acid template.
 25. The method ofclaim 17, wherein the at least second sequencing primer comprises atleast 2 contiguously annealing primers.
 26. The method of claim 17,wherein the at least second sequencing primer comprises at least 3contiguously annealing primers.
 27. The method of claim 24, wherein theplurality of contiguously annealing primers are 5 or 6 nucleotides inlength.
 28. The method of claim 24, wherein the plurality ofcontiguously annealing primers are independently selected from aplurality of primer sources comprising all possible primers of 5 or 6nucleotides in length.
 29. The method of claim 17, further comprising;repeating at least 10 times, the steps of selecting and introducingadditional sequencing primers to the at least first channel along withthe template nucleic acid based upon the nucleotide sequence of aprevious elongation reaction product, the additional sequencing primersreacting with the nucleic acid template to produce an additionalelongation reaction product.
 30. The method of claim 17, furthercomprising: repeating at least 20 times, the steps of selecting andintroducing additional sequencing primers to the at least first channelalong with the template nucleic acid based upon the nucleotide sequenceof a previous elongation reaction product, the additional sequencingprimers reacting with the nucleic acid template to produce an additionalelongation reaction product.
 31. The method of claim 17, furthercomprising: repeating at least 50 times, the steps of selecting andintroducing additional sequencing primers to the at least first channelalong with the template nucleic acid based upon the nucleotide sequenceof a previous elongation reaction product, the additional sequencingprimers reacting with the nucleic acid template to produce an additionalelongation reaction product.
 32. The method of claim 17, furthercomprising: repeating at least 100 times, the steps of selecting andintroducing additional sequencing primers to the at least first channelalong with the template nucleic acid based upon the nucleotide sequenceof a previous elongation reaction product, the additional sequencingprimers reacting with the nucleic acid template to produce an additionalelongation reaction product.
 33. The method of claim 17, furthercomprising: repeating the steps of selecting and introducing additionalsequencing primers to the at least first channel along with the templatenucleic acid based upon the nucleotide sequence of a previous elongationreaction product, the additional sequencing primers reacting with thenucleic acid template to produce an additional elongation reactionproduct, until a nucleotide sequence of substantially all of thetemplate nucleic acid is determined.
 34. The method of claim 1, whereinthe target nucleic acid, the first sequencing primer, the polymerase,the dNTPs, and the ddNTPs are mixed in a reservoir that is in fluidcommunication with a reagent introduction channel in the microfluidicdevice.
 35. The method of claim 1, wherein the target nucleic acid, thefirst sequencing primer, the polymerase, the dNTPs, and the ddNTPs aremixed in a reagent mixing channel in the microfluidic device.
 36. Themethod of claim 35, wherein the target nucleic acid, the firstsequencing primer, the polymerase, the dNTPs, and the ddNTPs areprovided in a plurality of different reservoirs that are in fluidcommunication with the reagent mixing channel, and are mixed in themicrofluidic device by simultaneously transporting the target nucleicacid, the first sequencing primer, the polymerase, the dNTPs, and theddNTPs into the reagent mixing channel.
 37. The method of claim 34,wherein the reagent introduction channel intersects a separationchannel, and the separating step comprises transporting thepolymerization products through the reagent introduction channel intothe separation channel, and separating the polymerization products. 38.The method of claim 35, wherein the reagent mixing channel intersects aseparation channel, and the separating step comprises transporting thepolymerization products from the reagent introduction channel into theseparation channel, and separating the polymerization products.
 39. Themethod of claim 37, wherein the polymerization products are separated inthe separation channel by electrophoresis.
 40. The method of claim 38,wherein the polymerization products are separated in the separationchannel by applying a voltage differential along a length of theseparation channel.
 41. The method of claim 37, wherein thepolymerization products are separated in the separation channel byelectrophoresis.
 42. The method of claim 37, wherein the polymerizationproducts are separated in the separation channel by applying a voltagedifferential along a length of the separation channel.
 43. The method ofclaims 37 or 38, wherein the polymerization products comprisefluorescent labels.
 44. The method of claim 1, wherein the providingstep comprises providing a plurality of sources of different sequencingprimers that includes a source of a first sequencing primer.
 45. Themethod of claim 44, wherein the plurality of sores of differentsequencing primers are provided separate from the microfluidic device,and the mixing step comprises delivering the first sequencing primerfrom the plurality of sources of sequencing primer to the microfluidicdevice to mix with the polymerase the dNTPs, and the ddNTPs.
 46. Themethod of claim 45, wherein the plurality of sources of sequencingprimers comprises at least about 70% of all primer sequences having thelength of the first primer sequence.
 47. The method of claim 1, whereinthe first sequencing primer comprises a plurality of separate primersthat are capable of contiguously annealing to the target nucleic acid.48. The method of claim 47, wherein each of the plurality of separateprimers that makes up the first sequencing primer has a length of fromabout 4 to about 10 nucleotides.
 49. The method of claim 1, wherein thefirst sequencing primer comprises at least three separate primers thatare capable of contiguously annealing to the target nucleic acid. 50.The method of claim 47, wherein the plurality of separate primers thatcomprise the first sequencing primer are selected from a plurality ofsources of primer sequences.
 51. The method of claim 50, wherein each ofthe separate primer sequences in the plurality of sources of primersequences has a length of from about 4 to about 10 nucleotides.
 52. Themethod of claim 50, wherein the plurality of sources of primer sequencesincludes at least 70% of all possible primer sequences of a givenlength, wherein the given length is from about 4 to bout 10 nucleotides.53. The method of claim 2, wherein the second sequencing primercomprises a plurality of separate primers that are capable ofcontiguously annealing to a portion of the first sequence of the targetnucleic acid.
 54. The method of claim 53, wherein each of the pluralityof separate primers that makes up the second sequencing primer has alength of from about 4 to about 10 nucleotides.
 55. The method of claim2, wherein the second sequencing primer comprises at least threeseparate primers that are capable of contiguously annealing to a portionof the first sequence of the target nucleic acid.
 56. The method ofclaim 55, wherein each of the separate primer sequences in the pluralityof separate primer sequences has a length of from about 4 to about 10nucleotides.
 57. The method of claim 55, wherein the plurality ofseparate primer sequences are selected from a plurality of sources ofprimer sequences.
 58. The method of claim 57, wherein the plurality ofsources of primer sequences includes at least 70% of all possible primersequences of a given length, wherein the given length is from about 4 tobout 10 nucleotides.
 59. The method of claim 2, further comprisingselecting a third primer sequence based upon the second sequence of thetarget nucleic acid, and mixing the third sequencing primer with thetarget nucleic acid under conditions to permit target dependentelongation of the third selected sequencing primer, thereby providing athird set of polymerization products which are separated by size in themicrofluidic device to provide a third sequence of the target nucleicacid.
 60. The method of claim 59, wherein the third sequencing primercomprises a plurality of separate primers that are capable ofcontiguously annealing to a portion of the second sequence of the targetnucleic acid.
 61. The method of claim 60, wherein each of the pluralityof primers that makes up the third sequencing primer has a length offrom about 4 to about 10 nucleotides.
 62. The method of claim 61,wherein the third sequencing primer comprises at least three separateprimers at are capable of contiguously annealing to a portion of thesecond sequence of target nucleic acid.
 63. The method of claim 62,wherein each of the primer sequences in the plurality of primersequences has a length of from about 4 to about 10 nucleotides.
 64. Themethod of claim 63, wherein the plurality of separate primer sequencesare selected from a plurality of sources of primer sequences.
 65. Themethod of claim 64, wherein the plurality of sources of primer sequencesincludes at least 70% of all possible primer sequences of a givenlength, wherein the given length is from about 4 to bout 10 nucleotides.